Methods for identifying cancer risk

ABSTRACT

Provided herein are tissue-specific differential methylated regions (T-DMRs) and cancer-related differential methylated regions (C-DMRs) and methods of use thereof. In one embodiment of the invention, there are provided methods of detecting a cell proliferative disorder by detecting altered methylation in one or more DMRs identified herein. In another embodiment of the invention, there are provided methods of determining clinical outcome by detecting altered methylation in one or more DMRs identified herein.

RELATED APPLICATION DATA

This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 61/118,169, filed Nov. 26, 2008, the entire content of which is incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made in part with government support under Grant Nos. P50HG003233 and R37CA54358 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to differentially methylated regions (DMRs) in the genome outside CpG islands, and more specifically to methods for detecting the presence of or a risk for a hyperproliferative disorder by detecting an alteration in methylation status of such DMRs.

2. Background Information

Epigenetics is the study of non-sequence information of chromosome DNA during cell division and differentiation. The molecular basis of epigenetics is complex and involves modifications of the activation or inactivation of certain genes. Additionally, the chromatin proteins associated with DNA may be activated or silenced. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime, but some epigenetic changes are inherited from one generation to the next.

One example of an epigenetic mechanism is DNA methylation (DNAm), a covalent modification of the nucleotide cytosine. In particular, it involves the addition of methyl groups to cytosine nucleotides in the DNA, to convert cytosine to 5-methylcytosine. DNA methylation plays an important role in determining whether some genes are expressed or not. Abnormal DNA methylation is one of the mechanisms underlying the changes observed with aging and development of many cancers.

Cancers have historically been linked to genetic changes such as DNA sequence mutations. Evidence now supports that a relatively large number of cancers originate, not from mutations, but from epigenetic changes such as inappropriate DNA methylation. In some cases, hypermethylation of DNA results the an inhibition of expression of critical genes, such as tumor suppressor genes or DNA repair genes, allowing cancers to develop. In other cases, hypomethylation of genes modulates expression, which contributes to the development of cancer.

Epigenetics has led to an epigenetic progenitor model of cancer that epigenetic alterations affecting tissue-specific division and differentiation are the predominant mechanism by which epigenetic changes cause cancer. In other words, it is believed that aberrant methylation patterns may play multiple roles in cancer, such as the silencing of tumor suppressor genes, and the over-expression of oncogenes.

Since the discovery of altered DNA methylation in human cancer, the focus has largely been on specific genes of interest and regions assumed to be important functionally, such as promoters and CpG islands, and there has not been a comprehensive genome-scale understanding of the relationship between DNA methylation loss and gain in cancer and in normal differentiation.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that some tissue-specific or cancer-related alterations in DNA methylation occur not only in promoters or CpG islands, but in sequences up to 2 kb distant from such CpG islands (such sequences are termed “CpG island shores”). In accordance with this discovery, there are provided herein differentially methylated regions (DMRs) and methods of use thereof.

In one embodiment of the invention, there are provided methods of diagnosis including detecting a cell proliferative disorder. The methods involve comparing the methylation status of one or more nucleic acid sequences in a sample from a subject suspected of having the disorder, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, to the methylation status of the one or more nucleic acid sequences in a sample from a corresponding normal tissue or individual not having a cell proliferative disorder, wherein an alteration in methylation status is indicative of a cell proliferative disorder.

In certain embodiments, the cell proliferative disorder is cancer. In some embodiments, the nucleic acid sequence is within a gene; alternatively, the nucleic acid sequence is upstream or downstream of a gene. In particular embodiments, the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof. In some embodiments the alteration in methylation status is hypomethylation; in other embodiments the alteration in methylation status is hypermethylation. In embodiments using more than one DMR, the alteration in methylation status of some may be hypomethylation, whereas others may be hypermethylation.

In another embodiment of the invention, there are provided methods of determining a clinical outcome. Such methods are accomplished by comparing the methylation status of one or more nucleic acid sequences in a sample from a subject prior to undergoing a therapeutic regimen for a disease or disorder, wherein the disease or disorder is associated with altered methylation of the one or more nucleic acid sequences, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, to the methylation status of the one or more nucleic acid sequences in a sample from the individual after the therapeutic regimen has been initiated, wherein change in methylation status is indicative a positive clinical outcome. In particular embodiments, the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof. In some embodiments the change in methylation status is hypomethylation; in other embodiments the change in methylation status is hypermethylation. In embodiments using more than one DMR, the change in methylation status of some may be hypomethylation, whereas others may be hypermethylation.

In another embodiment of the invention, there are provided methods for providing a methylation map of a region of genomic DNA by performing comprehensive high-throughput array-based relative methylation (CHARM) analysis on, for example, a sample of labeled, digested genomic DNA. In some embodiments, the method may further include bisulfate pyrosequencing of the genomic DNA, for example.

In still another embodiment of the invention, there are provided methods of detecting a methylation status profile of the nucleic acid of a cancer cell from a tumor or biological sample. Such methods include hybridizing labeled and digested nucleic acid of a cancer cell from a tumor or biological sample to a DNA microarray comprising at least 100 nucleic acid sequences, with the proviso that the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island and determining a pattern of methylation from the hybridizing of step a), thereby detecting a methylation profile. In particular embodiments, the method further includes comparing the methylation profile to a methylation profile from hybridization of the microarray with labeled and digested nucleic acid from control “normal” cells. In certain embodiments, the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof.

In yet another embodiment of the present invention, there are provided methods for prognosis of a cancer in a subject known to have or suspected of having a cancer associated with altered methylation of one or more nucleic acid sequences. The method includes obtaining a tissue sample or biological sample containing nucleic acid from a subject; and assaying the methylation status of one or more nucleic acid sequences, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island bodily fluid; wherein the presence of altered methylation in the sample from the subject, relative to a corresponding sample from a normal sample, is indicative that the subject is a good for therapy of cancer. In some embodiments, the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof.

In another embodiment of the invention, there is provided a plurality of nucleic acid sequences, wherein the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in cancer. In some embodiments, the nucleic acid sequence are selected from the group consisting of the DMR sequences as set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the MRPL36 gene, the MEST gene, the GATA-2 gene, and the RARES2 gene. In one aspect, the plurality is a microarray.

In another embodiment of the invention, there is provided a plurality of nucleic acid sequences, wherein the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in tissues derived from the three different embryonic lineages. In some embodiments, the plurality of nucleic acid sequences are selected from one or more of the sequences as set forth in FIG. 5 a, FIG. 5 b, Tables 1, 2, 4, 7, 9, 10, 12-14, and 17. In one aspect, the plurality is a microarray.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a plot of M value versus genomic location for brain, liver, and spleen (upper panel) and a plot of CpG density versus genomic location over the same region (lower panel) for a T-DMR located in a CpG island shore. FIG. 1B shows a plot of M value versus genomic location for brain, liver, spleen, normal colon, and colon tumor (upper panel) and a plot of CpG density versus genomic location over the same region (lower panel) for a C-DMR located in a CpG island shore that overlaps with a T-DMR.

FIG. 2 shows plots depicting the distribution of distance of T-DMRs and C-DMRs from CpG islands.

FIG. 3A shows a quantile-quantile plot depicting the number of sites of hypomethylation and hypermethylation in colon cancer. FIGS. 3B-J show box plots depicting the degree of DNA methylation of the indicated C-DMRs as measured by bisulfite pyrosequencing.

FIG. 4 shows a plot of differential gene expression versus differential methylation for brain and liver T-DMR.

FIG. 5 shows plots of the relative expression of hypermethylated T-DMR when treated with 5-aza-2′ deoxycytidine (FIG. 5A) or in a double knockout of DNA methyltransferase 1 and 3b (FIG. 5B).

FIG. 6 shows a clustering of human tissue samples using mouse T-DMRs.

FIGS. 7A and B show a clustering of normal tissue samples using C-DMRs.

FIGS. 8A-D show box plots of average ΔM values over all DMRs compared to randomly chosen regions and unmethylated control regions matched for length. FIG. 8E shows box plots of ΔM for pairwise comparison. FIG. 8F shows box plots of the average inter-individual standard deviation of the ΔM value for normal mucosa and colon tumors.

FIG. 9 shows plots of M values for CHARM probes located within a CGI (left plot) and outside a CGI (right plot).

FIGS. 10A-J show representative plots of 10 T-DMRs. The upper panels are plots of M value versus genomic location for brain, liver, and spleen. The middle panels provide the location of CpG dinucleotides with black tick marks on the x-axis. The lower panels provide gene annotation for the genomic region.

FIGS. 11A-C show plots of the distribution of distance of T-DMRs and C-DMRs from CpG islands using different FDR cutoff values (0.01, 0.05, and 0.10, for FIGS. 11A, 11B, and 11C, respectively.

FIGS. 12A-J show representative plots of 10 C-DMRs. The upper panels are plots of M value versus genomic location for brain, liver, spleen, normal colon, and colon cancer. The middle panels provide the location of CpG dinucleotides with black tick marks on the x-axis. The lower panels provide gene annotation for the genomic region.

FIGS. 13A-E shows box plots of bisulfite pyrosequencing confirming the prevalence of 5 hypermethylated C-DMR shores in a large set of colon tumor and normal mucosa samples. Box-plots represent DNA methylation level measured using bisulfite pyrosequencing. A, distal-less homeobox 5 (DLX5); B, leucine rich repeat and fibronectin type III domain containing 5 (LRFN5); C, homeobox A3 (HOXA3); D, SLIT and NTRK-like family, member 1 (SLITRK1); E, FEZ family zinc finger 2 (FEZF2), (n) equals the number of samples analyzed by pyrosequencing.

FIGS. 14A-D shows box plots of bisulfite pyrosequencing confirming the prevalence of 4 hypomethylated C-DMR shores in a large set of colon tumor and normal mucosa samples. Box-plots represent DNA methylation level measured using bisulfite pyrosequencing. A, transmembrane protein 14A (TMEM14A); B, glutamate-rich 1 (ERICH1); C, family with sequence similarity 70, member B (FAM70C); D, prostate transmembrane protein, androgen induced 1 (TMEPAI), (n) equals the number of samples analyzed by pyrosequencing.

FIG. 15 shows a plot of log (base 2) ratios of colon tumor to normal expression against delta M values for colon tumor and normal DNAm.

FIG. 16 shows a plot of average delta M values stratified into T-DMRs, hypermethylated C-DMRs and hypomethylated C-DMRs. T-DMRs were further stratified according to brain versus liver, brain versus spleen, and liver versus spleen pair-wise comparisons. The box-plots represent absolute values of the delta Ms.

FIG. 17 shows a plot the average inter-individual standard deviation of the M-values for C-DMRs. The box-plots represent these values for normal colon mucosa and colon tumors.

FIG. 18A shows a schematic overview of the PIP5K1A gene locus. C-DMR represents the genomic region that is hypomethylated in colon tumors as compared to normals.

FIG. 18B shows a photograph of a gel in which the arrow indicates a 220 bp fragment present in 3 colon tumors but not normal mucosa from the same individual amplified by the Sp3 primer and the 5′ RACE anchor primer.

FIG. 19 shows an example of C-DMR that is not adjacent to a conventionally defined CpG island. The upper panel is a plot of M value versus genomic location for brain, liver, spleen, normal colon, colon tumor. The middle panel provides the location of CpG dinucleotides with black tick marks on the x-axis. The lower panel provides gene annotation for the genomic region.

FIG. 20 shows a plot of hierarchical clustering. Columns represent individual samples, and rows represent regions corresponding to mouse T-DMRs. The heatmap displays M values, with red being more methylated and blue less.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that some tissue-specific or cancer-related alterations in DNA methylation occur not only in promoters or CpG islands, but in sequences up to 2 kb distant (termed “CpG island shores”). In accordance with this discovery, there are provided herein tissue-specific differential methylated regions (T-DMRs) and cancer-related differential methylated regions (C-DMRs) and methods of use thereof. Accordingly, in one embodiment of the invention, there are provided methods of detecting a cell proliferative disorder. The methods involve comparing the methylation status of one or more nucleic acid sequences in a sample from a subject suspected of having the disorder, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, to the methylation status of the one or more nucleic acid sequences in a sample from a corresponding normal tissue or individual not having a cell proliferative disorder, wherein an alteration in methylation status is indicative of a cell proliferative disorder. In some embodiments the alteration in methylation status is hypomethylation; in other embodiments the alteration in methylation status is hypermethylation. In embodiments using more than one DMR, the alteration in methylation status of some may be hypomethylation, whereas others may be hypermethylation.

In some embodiments methylation status is converted to an M value. As used herein an M value, can be a log ratio of intensities from total (Cy3) and McrBC-fractionated DNA (Cy5): positive and negative M values are quantitatively associated with methylated and unmethylated sites, respectively.

Hypomethylation of a DMR is present when there is a measurable decrease in methylation of the DMR. In some embodiments, a DMR can be determined to be hypomethylated when less than 50% of the methylation sites analyzed are not methylated. Hypermethylation of a DMR is present when there is a measurable increase in methylation of the DMR. In some embodiments, a DMR can be determined to be hypermethylated when more than 50% of the methylation sites analyzed are methylated. Methods for determining methylation states are provided herein and are known in the art. In some embodiments methylation status is converted to an M value. As used herein an M value, can be a log ratio of intensities from total (Cy3) and McrBC-fractionated DNA (Cy5): positive and negative M values are quantitatively associated with methylated and unmethylated sites, respectively. M values are calculated as described in the Examples. In some embodiments, M values which range from ±0.5 to 0.5 represent unmethylated sites as defined by the control probes, and values from 0.5 to 1.5 represent baseline levels of methylation.

In particular embodiments, the one or more nucleic acid sequence is selected from the C-DMRs provided herein. In one aspect, the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof. In some embodiments, the nucleic acid sequence is within a gene; alternatively, the nucleic acid sequence is upstream or downstream of a gene.

In particular embodiments, the one or more nucleic acid sequence is selected from the T-DMRs provided herein. In one aspect, the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in FIG. 5 a, FIG. 5 b, Tables 1, 2, 4, 7, 9, 10, 12-14, and 17. Such T-DMRs may be used to distinguish between the tissue types representing the three embryonic lineages: endodermal, mesodermal, and ectodermal.

The biological sample can be virtually any biological sample, particularly a sample that contains RNA or DNA from the subject. The biological sample can be a tissue sample which contains about 1 to about 10,000,000, about 1000 to about 10,000,000, or about 1,000,000 to about 10,000,000 somatic cells. However, it is possible to obtain samples that contain smaller numbers of cells, even a single cell in embodiments that utilize an amplification protocol such as PCR. The sample need not contain any intact cells, so long as it contains sufficient biological material (e.g., protein or genetic material, such as RNA or DNA) to assess methylation status of the one or more DMRs.

In some embodiments, a biological or tissue sample can be drawn from any tissue that is susceptible to cancer. A biological or tissue sample may be obtained by surgery, biopsy, swab, stool, or other collection method. In some embodiments, the sample is derived from blood, plasma, serum, lymph, nerve-cell containing tissue, cerebrospinal fluid, biopsy material, tumor tissue, bone marrow, nervous tissue, skin, hair, tears, fetal material, amniocentesis material, uterine tissue, saliva, feces, or sperm. In particular embodiments, the biological sample for methods of the present invention can be, for example, a sample from colorectal tissue, or in certain embodiments, can be a blood sample, or a fraction of a blood sample such as a peripheral blood lymphocyte (PBL) fraction. Methods for isolating PBLs from whole blood are well known in the art. In addition, it is possible to use a blood sample and enrich the small amount of circulating cells from a tissue of interest, e.g., colon, breast, lung, prostate, head and neck, etc. using a method known in the art.

As disclosed above, the biological sample can be a blood sample. The blood sample can be obtained using methods known in the art, such as finger prick or phlebotomy. Suitably, the blood sample is approximately 0.1 to 20 ml, or alternatively approximately 1 to 15 ml with the volume of blood being approximately 10 ml.

Accordingly, in one embodiment, the identified cancer risk is for colorectal cancer, and the biological sample is a tissue sample obtained from the colon, blood, or a stool sample. In another embodiment, the identified cancer risk is for stomach cancer or esophageal cancer, and the tissue may be obtained by endoscopic biopsy or aspiration, or stool sample or saliva sample. In another embodiment, the identified cancer risk is esophageal cancer, and the tissue is obtained by endoscopic biopsy, aspiration, or oral or saliva sample. In another embodiment, the identified cancer risk is leukemia/lymphoma and the tissue sample is blood.

In the present invention, the subject is typically a human but also can be any mammal, including, but not limited to, a dog, cat, rabbit, cow, bird, rat, horse, pig, or monkey.

As mentioned above, for certain embodiments of the present invention, the method is performed as part of a regular checkup. Therefore, for these methods the subject has not been diagnosed with cancer, and typically for these present embodiments it is not known that a subject has a hyperproliferative disorder, such as a cancer.

Methods of the present invention identify a risk of developing cancer for a subject. A cancer can include, but is not limited to, colorectal cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, lung cancer, prostate cancer, uterine cancer, breast cancer, skin cancer, endocrine cancer, urinary cancer, pancreas cancer, other gastrointestinal cancer, ovarian cancer, cervical cancer, head cancer, neck cancer, and adenomas. In one aspect, the cancer is colorectal cancer.

A hyperproliferative disorder includes, but is not limited to, neoplasms located in the following: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital. In certain embodiments, the hyperproliferative disorder is a cancer. In one aspect, the cancer is colorectal cancer.

In another embodiment, the present invention provides a method for managing health of a subject. The method includes performing the method for identifying an increased risk of developing cancer discussed above and performing a traditional cancer detection method. For example a traditional cancer detection method can be performed if the method for identifying cancer risk indicates that the subject is at an increased risk for developing cancer. Many traditional cancer detection methods are known and can be included in this aspect of the invention. The traditional cancer detection method can include, for example, one or more of chest X-ray, carcinoembryonic antigen (CEA) level determination, colorectal examination, endoscopic examination, MRI, CAT scanning, or other imaging such as gallium scanning, and barium imaging, and sigmoidoscopy/colonoscopy, a breast exam, or a prostate specific antigen (PSA) assay.

Numerous methods for analyzing methylation status of a gene are known in the art and can be used in the methods of the present invention to identify either hypomethylation or hypermethylation of the one or more DMRs. In some embodiments, the determining of methylation status is performed by one or more techniques selected from the group consisting of a nucleic acid amplification, polymerase chain reaction (PCR), methylation specific PCR, bisulfite pyrosequenceing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, microarray technology, and proteomics. As illustrated in the Examples herein, analysis of methylation can be performed by bisulfite genomic sequencing. Bisulfite treatment modifies DNA converting unmethylated, but not methylated, cytosines to uracil. Bisulfite treatment can be carried out using the METHYLEASY bisulfite modification kit (Human Genetic Signatures).

In some embodiments, bisulfite pyrosequencing, which is a sequencing-based analysis of DNA methylation that quantitatively measures multiple, consecutive CpG sites individually with high accuracy and reproducibility, may be used. Exemplary primers for such analysis are set forth in Table 8.

It will be recognized that depending on the site bound by the primer and the direction of extension from a primer, that the primers listed above can be used in different pairs. Furthermore, it will be recognized that additional primers can be identified within the DMRs, especially primers that allow analysis of the same methylation sites as those analyzed with primers that correspond to the primers disclosed herein.

Altered methylation can be identified by identifying a detectable difference in methylation. For example, hypomethylation can be determined by identifying whether after bisulfite treatment a uracil or a cytosine is present a particular location. If uracil is present after bisulfite treatment, then the residue is unmethylated. Hypomethylation is present when there is a measurable decrease in methylation.

In an alternative embodiment, the method for analyzing methylation of the DMR can include amplification using a primer pair specific for methylated residues within a DMR. In these embodiments, selective hybridization or binding of at least one of the primers is dependent on the methylation state of the target DNA sequence (Herman et al., Proc. Natl. Acad. Sci. USA, 93:9821 (1996)). For example, the amplification reaction can be preceded by bisulfite treatment, and the primers can selectively hybridize to target sequences in a manner that is dependent on bisulfite treatment. For example, one primer can selectively bind to a target sequence only when one or more base of the target sequence is altered by bisulfite treatment, thereby being specific for a methylated target sequence.

Other methods are known in the art for determining methylation status of a DMR, including, but not limited to, array-based methylation analysis and Southern blot analysis.

Methods using an amplification reaction, for example methods above for detecting hypomethylation or hyprmethylation of one or more DMRs, can utilize a real-time detection amplification procedure. For example, the method can utilize molecular beacon technology (Tyagi S., et al., Nature Biotechnology, 14: 303 (1996)) or Taqman™ technology (Holland, P. M., et al., Proc. Natl. Acad. Sci. USA, 88:7276 (1991)).

Also methyl light (Trinh B N, Long T I, Laird P W. DNA methylation analysis by MethyLight technology, Methods, 25(4):456-62 (2001), incorporated herein in its entirety by reference), Methyl Heavy (Epigenomics, Berlin, Germany), or SNuPE (single nucleotide primer extension) (See e.g., Watson D., et al., Genet Res. 75(3):269-74 (2000)). Can be used in the methods of the present invention related to identifying altered methylation of DMRs.

As used herein, the term “selective hybridization” or “selectively hybridize” refers to hybridization under moderately stringent or highly stringent physiological conditions, which can distinguish related nucleotide sequences from unrelated nucleotide sequences.

As known in the art, in nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, relative GC:AT content), and nucleic acid type, i.e., whether the oligonucleotide or the target nucleic acid sequence is DNA or RNA, can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Methods for selecting appropriate stringency conditions can be determined empirically or estimated using various formulas, and are well known in the art (see, for example, Sambrook et al., supra, 1989).

An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps listed.

The degree of methylation in the DNA associated with the DMRs being assessed, may be measured by fluorescent in situ hybridization (FISH) by means of probes which identify and differentiate between genomic DNAs, associated with the DMRs being assessed, which exhibit different degrees of DNA methylation. FISH is described in the Human chromosomes: principles and techniques (Editors, Ram S. Verma, Arvind Babu Verma, Ram S.) 2nd ed., New York: McGraw-Hill, 1995, and de Capoa A., Di Leandro M., Grappelli C., Menendez F., Poggesi I., Giancotti P., Marotta, M. R., Spano A., Rocchi M., Archidiacono N., Niveleau A. Computer-assisted analysis of methylation status of individual interphase nuclei in human cultured cells. Cytometry. 31:85-92, 1998 which is incorporated herein by reference. In this case, the biological sample will typically be any which contains sufficient whole cells or nuclei to perform short term culture. Usually, the sample will be a tissue sample that contains 10 to 10,000, or, for example, 100 to 10,000, whole somatic cells.

Additionally, as mentioned above, methyl light, methyl heavy, and array-based methylation analysis can be performed, by using bisulfite treated DNA that is then PCR-amplified, against microarrays of oligonucleotide target sequences with the various forms corresponding to unmethylated and methylated DNA.

The term “nucleic acid molecule” is used broadly herein to mean a sequence of deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the term “nucleic acid molecule” is meant to include DNA and RNA, which can be single stranded or double stranded, as well as DNA/RNA hybrids. Furthermore, the term “nucleic acid molecule” as used herein includes naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR), and, in various embodiments, can contain nucleotide analogs or a backbone bond other than a phosphodiester bond.

The terms “polynucleotide” and “oligonucleotide” also are used herein to refer to nucleic acid molecules. Although no specific distinction from each other or from “nucleic acid molecule” is intended by the use of these terms, the term “polynucleotide” is used generally in reference to a nucleic acid molecule that encodes a polypeptide, or a peptide portion thereof, whereas the term “oligonucleotide” is used generally in reference to a nucleotide sequence useful as a probe, a PCR primer, an antisense molecule, or the like. Of course, it will be recognized that an “oligonucleotide” also can encode a peptide. As such, the different terms are used primarily for convenience of discussion.

A polynucleotide or oligonucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template

In another aspect, the present invention includes kits that are useful for carrying out the methods of the present invention. The components contained in the kit depend on a number of factors, including: the particular analytical technique used to detect methylation or measure the degree of methylation or a change in methylation, and the one or more DMRs is being assayed for methylation status.

Accordingly, the present invention provides a kit for determining a methylation status of one or more differentially methylated region (DMR) of the invention. In some embodiments, the one or more T-DMRs are selected from one or more of the sequences as set forth in Tables 1-4, 6, 7, 9, 11, 14-16, the MRPL36 gene, the MEST gene, the GATA-2 gene, and the RARES2 gene. In another embodiment, the one or more T-DMRs are selected from one or more of the sequences as set forth in FIG. 5 a, FIG. 5 b, Tables 1, 2, 4, 7, 9, 10, 12-14, and 17. The kit includes an oligonucleotide probe, primer, or primer pair, or combination thereof for carrying out a method for detecting hypomethylation, as discussed above. For example, the probe, primer, or primer pair, can be capable of selectively hybridizing to the DMR either with or without prior bisulfite treatment of the DMR. The kit can further include one or more detectable labels.

The kit can also include a plurality of oligonucleotide probes, primers, or primer pairs, or combinations thereof, capable of selectively hybridizing to the DMR with or without prior bisulfite treatment of the DMR. The kit can include an oligonucleotide primer pair that hybridizes under stringent conditions to all or a portion of the DMR only after bisulfite treatment. In one aspect, the kit can provide reagents for bisulfite pyrosequencing including one or more primer pairs set forth in Table 8. The kit can include instructions on using kit components to identify, for example, the presence of cancer or an increased risk of developing cancer.

The studies provided herein focused on three key questions. First, where are DNA methylation changes that distinguish tissue types? Taking a comprehensive genome-wide approach, three normal tissue types representing the three embryonic lineages—liver (endodermal), spleen (mesodermal) and brain (ectodermal)—obtained from five autopsies were examined. A difference from previous methylation studies of tissues, aside from the genome-wide design herein, is that in the present studies, tissues were obtained from the same individual, thus controlling for potential interindividual variability. Second, where are DNAm alterations in cancer, and what is the balance between hypomethylation and hypermethylation? For this purpose, 13 colorectal cancers and matched normal mucosa from the subjects were examined. Third, what is the functional role of these methylation changes? To this end, a comparative epigenomics study of tissue methylation in the mouse, as well as gene expression analyses were carried out.

To examine DNAm on a genome-wide scale, comprehensive high-throughput array-based relative methylation (CHARM) analysis, which is a microarray-based method agnostic to preconceptions about DNAm, including location relative to genes and CpG content was carried out. The resulting quantitative measurements of DNAm, denoted with M, are log ratios of intensities from total (Cy3) and McrBC-fractionated DNA (Cy5): positive and negative M values are quantitatively associated with methylated and unmethylated sites, respectively. For each sample, ˜4.6 million CpG sites across the genome were analyzed using a custom-designed NimbleGen HD2 microarray, including all of the classically defined CpG islands as well as all nonrepetitive lower CpG density genomic regions of the genome. 4,500 control probes were included to standardize these M values so that unmethylated regions were associated, on average, with values of 0. CHARM is 100% specific at 90% sensitivity for known methylation marks identified by other methods (for example, in promoters) and includes the approximately half of the genome not identified by conventional region preselection. The CHARM results were also extensively corroborated by quantitative bisulfite pyrosequencing analysis.

Provided herein is a genome-wide analysis of DNA methylation addressing variation among normal tissue types, variation between cancer and normal, and variation between human and mouse, revealing several surprising relationships among these three types of epigenetic variation, supported by extensive bisulfite pyrosequencing and functional analysis. First, most tissue-specific DNAm was found to occur, not at CpG islands, but at CpG island shores (sequences up to 2 kb distant from CpG islands). The identification of these regions opens the door to functional studies, such as those investigating the mechanism of targeting DNAm to these regions and the role of differential methylation of shores. Supporting a functional role for shores, gene expression was closely linked to T-DMR and C-DMR methylation, particularly for switches from ‘none’ to ‘some’ methylation. The relationship between shore methylation and gene expression was confirmed by 5-aza-2′-deoxycytidine and DNA methyltransferase knockout experiments altering expression of the same genes. Another mechanism for shores supported by this study is regulation of alternative transcripts, supported by mapping and RACE experiments.

Although 76% of T-DMRs identified herein were in CpG island shores, at least for the three tissues examined here, 24% were not adjacent to conventionally defined CpG islands. However, many of these regions were nevertheless shores of CpG-enriched sequences (for an example, see FIG. 13). We are currently developing a novel algorithm for CpG island definition based on hidden Markov modeling that will likely increase the fraction of T-DMRs in CpG island shores. The ‘CpG clusters’ recently identified (Glass et al., Nucleic Acids Res 35:6798-6807, 2007) are not CpG island shores (only 4% of shore DMRs map to them), although the shores of these clusters, like the shores of CpG islands, are enriched for DMRs. Note that the variation in DNAm is still not within the dense CpG regions as defined by any of these definitions but in CpG shores.

A second key finding of the studies provided herein is that T-DMRs are highly conserved between human and mouse, and the methylation itself is sufficiently conserved to completely discriminate tissue types regardless of species of origin. This was true even for T-DMRs located >2 kb from transcriptional start sites. The incorporation of epigenetic data, such as DNAm, in evolutionary studies as done here, should greatly enhance the identification of conserved elements that regulate differentiation. Greater DNAm heterogeneity was found in human than in mouse (at least in an inbred strain), even for DMRs located >2 kb from a gene promoter. While not wishing to be bound to any particular theory, this result suggests that the conservation of DNAm between human and mouse may have a strong genetic basis, consistent with a greater degree of tissue DNAm homogeneity in the inbred mouse strain.

A third key finding of the studies provided herein is that most cancer-related changes in DNAm, that is, C-DMRs, at least for colon cancer, correspond to T-DMRs, and that these changes are similarly divided between hypomethylation and hypermethylation and also involve CpG island shores. Thus, epigenetic changes in cancer largely involve the same DMRs as epigenetic changes in normal differentiation. These results have important implications for studies such as the Cancer Genome Atlas, in that most altered DNA methylation in cancer does not involve CpG islands, and thus these studies would benefit from analysis of CpG island shores. Similarly, high-throughput sequencing efforts based on reduced representation analysis of CpG islands per se are unlikely to identify most DNAm variation in normal tissues or in cancer.

Finally, GO annotation analysis suggests that DNAm changes in cancer reflect development and pluripotency-associated genes, and differentiated cellular functions for lineages other than the colon. These data are consistent with the epigenetic progenitor model of cancer (Feinberg et al., Nat Rev Genet. 7:21-33, 2006), which proposes that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer. The genes identified in the studies provided herein will themselves be of considerable interest for further study, as will be the potential regulatory regions that did not lie in close proximity to annotated genes.

The following example is provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE The Human Colon Cancer Methylome Shows Similar Hypo- and Hypermethylation at Conserved Tissue-Specific CpG Island Shores

It has been shown that alterations in DNA methylation (DNAm) occur in cancer, including hypomethylation of oncogenes and hypermethylation of tumor suppressor genes. However, most studies of cancer methylation have assumed that functionally important DNAm will occur in promoters, and that most DNAm changes in cancer occur in CpG islands. This example illustrates that most methylation alterations in colon cancer occur not in promoters, and also not in CpG islands, but in sequences up to 2 kb distant, which are termed herein ‘CpG island shores’. CpG island shore methylation was strongly related to gene expression, and it was highly conserved in mouse, discriminating tissue types regardless of species of origin. There was a notable overlap (45-65%) of the locations of colon cancer-related methylation changes with those that distinguished normal tissues, with hypermethylation enriched closer to the associated CpG islands, and hypomethylation enriched further from the associated CpG island and resembling that of non-colon normal tissues. Thus, methylation changes in cancer are at sites that vary normally in tissue differentiation, consistent with the epigenetic progenitor model of cancer, which proposes that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer.

Samples. Snap-frozen colon tumors and dissected normal mucosa were obtained from the same subjects. For the tissue studies, human postmortem brain, liver and spleen tissues, from the same individual, were obtained.

Genomic DNA isolation and McrBC fractionation. Genomic DNA isolation was carried out using the MasterPure DNA purification kit (Epicentre) as recommended by the manufacturer. For each sample, 5 m of genomic DNA was digested, fractionated, labeled and hybridized to a CHARM microarray (Irizarrry et al., Genome Res 18:771-9, 2008).

CHARM microarray design. CHARM microarrays were prepared as previously described (Irizarrry et al., Genome Res 18:771-9, 2008) and additionally included a set of 4,500 probes, totaling 148,500 base pairs across 30 genomic regions as controls. As these control probes represent genomic regions without CpG sites and hence cannot be methylated, they were used to normalize and standardize array data. The observed M values were standardized so that the average in the control regions was 0. Therefore, M values of 0 for other probes on the array were associated with no methylation.

CHARM DNA methylation analysis. McrBC fractionation was conducted followed by CHARM array hybridization for all human tissue samples as previously described (Irizarrry et al., Genome Res 18:771-9, 2008). For each probe, average M values were computed across the five samples in each tissue type. Differential methylation was quantified for each pairwise tissue comparison by the difference of averaged M values (ΔM). Replicates were used to estimate probe-specific s.d., which provided standard errors (s.e.m.) for ΔM. z scores (ΔM/s.e.m.(:ΔM)) were calculated and grouped contiguous statistically significant values into regions. Because millions of z scores were examined, statistical confidence calculation needed to account for multiple comparisons. Therefore false discovery rates (FDR) were computed and a list with an FDR of 5% was reported. Statistical significance of the regions was assessed as described below. C-DMRs were determined using the same procedure described above with the following exception: because greater heterogeneity was observed in the cancer samples (FIG. 8 f), ΔM was not divided by the standard errors, as this would penalize regions of highly variable M values. For all expression microarray analysis, RMA was used for processing and then the samples were averaged in each tissue, and the difference (equivalent to average log ratio) computed. Mouse T-DMRs were determined using the same statistical procedures as described above for the T-DMRs and were then mapped to the human genome using the UCSC liftOver tool. To correct for possible ‘array’ effects, each T-DMR was standardized by subtracting the mean of M across all samples within a species and divided by s.d. across all samples within a species. A list of all mouse T-DMRs is provided in Table 17. Overlap of C-DMRs with T-DMRs was determined by adding the number of regions.

Statistical significance of DMRs. Contiguous regions composed of probes with z scores associated with P values smaller than 0.001 were grouped into regions. The area of each region (length multiplied by ΔM) was used to define statistical significance. A permutation test was used to form a null distribution for these areas and the empirical Bayes approach described. The effect of fragment length on M values observed using CHARM was tested by computing the expected DNA fragment size based on McrBC recognition sites. Next, the ΔM values were stratified for each probe, from the colon tumor and normal mucosa comparison, by fragment size. The results showed no relationship between fragment size and ΔM.

Bisulfite pyrosequencing. Isolation of genomic DNA for all bisulfite pyrosequencing validation was done using the MasterPure DNA purification kit (Epicentre) as recommended by the manufacturer. For validation of shore regions, 1 μg of genomic DNA from each sample was bisulfite-treated using an EpiTect kit (Qiagen) according to the manufacturer's specifications. Converted genomic DNA was PCR-amplified using unbiased nested primers and carried out quantitative pyrosequencing using a PSQ HS96 (Biotage) to determine percentage methylation at each CpG site. Primer sequences and annealing temperatures are provided in Table 8. For bisulfite pyrosequencing of C-DMRs in 34-65 colon tumor and 30-61 normal mucosa samples, 500 ng of genomic DNA was bisulfite-treated using the EZ-96 DNA Methylation Gold kit (Zymo Research) as specified by the manufacturer. Converted genomic DNA was PCR-amplified using unbiased nested primers followed by pyrosequencing using a PSQ HS96 (Biotage). Bisulfite pyrosequencing was done as previously described (Tort et al., Nat Protocols 2:2265-75, 2007). Percent methylation was determined at each CpG site using the Q-CpG methylation software (Biotage). Table 6 provides the genomic location of CpG sites measured in the CpG shore and associated CpG island bisulfite pyrosequencing assays. Genomic coordinates for all CpG sites measured in the set of ˜50 colon tumor and normal samples are provided in Methods online. The genomic coordinates for CpG sites measured in colon tumor and normal samples representing DLX5C, ERICH1, FAM70B, SLITRK1, FEZF2, LRFN5, TMEM14A, TMEPAI, and HOXA3 are chr7:96493826,96493847; chr8: 847868,847870; chr13:113615615; chr13:83352935; chr3:62335457,62335482,62335504; chr14:411-48241,41148246,41148252; chr6:52638087; chr20:55707264; and chr7:27130446,27130448,27130450, respectively. Primer sequences and annealing temperatures for all bisulfite pyrosequencing reactions are provided in Table 7.

Total RNA isolation. Total RNA was isolated for Affymetrix microarray analysis from all human tissues using the RNEASY Mini kit (Qiagen) as specified by the manufacturer. All samples were DNase treated using the on-column DNase digestion kit (Qiagen) as recommended. Total RNA concentration was measured and RNA quality was determined by using an RNA 6000 Nano Lab chip kit and running the chip on a 2100 Bioanalyzer (Agilent).

Affymetrix microarray expression analysis. Genome-wide transcriptional analysis was done on a total of five liver and five brain samples from the same individuals using Affymetrix U133A GeneChip microarrays. The raw microarray gene expression data was obtained for the brain and liver tissue from The Stanley Medical Research Institute (SMRI) online genomics database (see URLs section below). The five individuals selected were unaffected controls from the SMRI Array collection. Genome-wide transcriptional profiling was also carried out on four colon tumor and four matched normal mucosa using Affymetrix U133 Plus 2.0 microarrays. One microgram of high-quality total RNA was amplified, labeled and hybridized according to the manufacturer's (Affymetrix) specifications and data was normalized as previously described (Irizarry et al. Biostatistics 4:249-64, 2003; and Bolstad et al., Bioinformatics 19:185-93, 1999).

Quantitative real-time PCR. Quantitative real time PCR was performed on high quality total RNA samples, determined using Agilent Bioanalyzer and RNA Nano 6000 chips, using pre-optimized Taqman assays (Applied Biosystems). A summary of assay identification numbers can be found in Table 9. Total RNA was prepared for quantitative real-time PCR using the Trizol method (Invitrogen) for FZD3, RBM38, NDN and SEMA3C and the RNEASY mini kit (Qiagen) was used to isolate total RNA for ZNF804A, CHRM2 and NQO1. cDNA was prepared for quantitative real-time PCR using the QUANTITECT RT kit (Qiagen) with Turbo DNase to eliminate genomic DNA. TaqMan assays (Applied Biosystems) were used to determine relative gene expression and analyzed experiments on a 7900HT detection system. Taqman assays (Applied Biosystems) were used to determine relative gene expression and experiments were analyzed on a 7900HT detection system. Human ACTB was used as an endogenous control. Relative expression differences were calculated using the ΔΔCt method (Livak and Schmittgen, Methods 25:402-8, 2001).

5′ RACE PCR. 5′ RACE experiments were done using a second generation RACE kit (Roche Applied Science) as specified by the manufacturer's protocol. RACE PCR products were directly sequenced with 3100 Genetic Analyzer (AB Applied Biosystems). The sequences for our gene specific primers are: PIP5K1A cDNA synthesis (PIP5K1A-Sp1): TCCTGAGGAATCAACACTTC; first round PIP5K1A primer (PIP5K1A-Sp2): CAGATGCCATGGGTCTCTTG; second round PIP5K1A primer (PIP5K1A-Sp3): ACGTCGAGCCGGCTCCTGGA.

Soft agar assay for colony growth. HeLa and HCT116 cell lines were purchased from American Type Culture Collection (ATCC) and cultured using media and conditions recommended by the supplier. Cells were transfected with sequence verified full-length cDNAs of NQO1, ZNF804A, and CHRM2, and empty expression plasmid constructs obtained from Open Biosystems (Huntsville, Ala.) using Fugene (Roche) following supplied protocols. After 48 hours cells were harvested by trypsin treatment and resuspended in quadruplets at 5000 cells/35 mm well in 0.35% agar overlaid over 0.5% agar. The culture media contained 1×DMEM, 10% fetal bovine serum, 100 units/ml penicillin/streptomycin. Cells were incubated in a humidified CO₂ incubator (37° C., 5% CO₂) for 17 days followed by staining with 0.005% Crystal Violet for 2 hours. Colonies were counted under a light microscope.

GO annotation. GO annotation was analyzed using the Bioconductor Gostats package to find enriched categories (P<0.01).

URLs. Complete set of T-DMR plots is on the internet at rafalab.jhsph.edu/t-dmr3000.pdf; complete set of C DMR plots is on the internet at rafalab.jhsph.edu/c-dmr-all.pdf. The Stanley Medical Research Institute (SMRI) online genomics database is available at stanleygenomics.org.

Accession codes. NCBI GEO: Gene expression microarray data was submitted under accession number GSE13471.

Most tissue-specific DNAm occurs in ‘CpG island shores.’ Because CHARM is not biased for CpG island or promoter sequences, objective data on tissue-specific methylation could be obtained. 16,379 tissue differential methylation regions (T-DMRs), defined as regions with M values for one tissue consistently different than that for the others at a false discovery rate (FDR) of 5% (see Methods) were identified. The median size of a T-DMR was 255 bp. Previous studies of tissue- or cancer-specific DNAm have focused on promoters and/or CpG islands, which have been defined as regions with a GC fraction greater than 0.5 and an observed-to-expected ratio of CpG greater than 0.6 (Feinberg, A. P. & Tycko, B., Nat. Rev. Cancer 4, 143-153, 2004; and Gardiner-Garden, M. & Frommer, M., J. Mol. Biol. 196, 261-282, 1987). It has previously been reported that the degree of differences in DNAm of promoters in somatic cells is relatively low in conventionally defined CpG islands and higher at promoters with intermediate CpG density. Two recent studies identified a relatively small fraction, 4-8%, of CpG islands with tissue-specific methylation. It was also found herein that DNAm variation is uncommon in CpG islands (FIG. 9).

The genome-wide approach of CHARM also enabled the finding of an unexpected physical relationship between CpG islands and DNAm variation, namely that 76% of T-DMRs were located within 2 kb of islands in regions denoted herein as ‘CpG island shores.’ For example, for the T-DMR in the PRTFDC1 gene, which encodes a brain-specific phosphoribosyltransferase that is relatively hypomethylated in the brain, the spreading of M values among the tissues begins ˜200 by from the CpG island and at a point where the CpG density associated with the island has fallen to 1/10 the density in the island itself (FIG. 1). The association of T-DMRs with CpG island shores was not due to an arbitrary definition of CpG islands but to a true association of these DNAm differences near but not in the regions of dense CpG content (Table 10 describes some of the identified T-DMR regions. The complete set of T-DMRs is available on the Nature Genetics website (nature.com/naturegenetics) see “Supplementary Data 1” in the Supplementary Information for Irizarry et al., Nature Genetics 41(2):178-186). Plots similar to those in FIG. 1 for 10 of the T-DMRs are provided in FIG. 10A-J; the complete set of T-DMRs plots, ordered by statistical significance, are available online at rafalab.jhsph.edu/t-dmr3000.pdf). The distribution of T-DMRs by distance from the respective islands showed that DNAm variation is distributed over a ˜2 kb shore, and that although CpG islands are enriched on the arrays, because of their high CpG content (33% of CHARM probes are in islands), only 6% of T-DMRs are in islands, compared to 76% in shores; an additional 18% of T-DMRs were located greater than 2 kb from the respective islands (FIG. 2). The localization of T-DMRs also occurred largely outside of promoters (96%), as CpG islands are localized primarily within promoters. Furthermore, more than half (52%) of T-DMRs were greater than 2 kb from the nearest annotated gene. The distribution of the distance to islands remained essentially unchanged when we used FDR cutoffs of 0.01, 0.05 and 0.10.

The array-based result that the differential methylation was in CpG island shores rather than in the associated islands was confirmed by carrying out bisulfite pyrosequencing analysis on over 100 CpG sites in the islands and shores associated with four genes, three T-DMRs and one cancer differential methylation region. At all 101 sites, the DMR was confirmed to lie within the shore rather than the island (Table 1). For example, PCDH9, which encodes a brain-specific protocadherin, was relatively hypomethylated in the brain at all 6 sites examined in the CpG island shore but unmethylated in both brain and spleen at all 18 sites examined in the associated island (Table 1). Differential methylation of an additional four CpG island shores was also confirmed by bisulfite pyrosequencing of 39 total CpG sites, and all showed statistically significant differences in DNAm (P<0.05) (Table 2). These data verify the sensitivity of CHARM for detecting subtle differences in DNAm. Furthermore, they confirm that most normal differential methylation takes place at CpG island shores.

FIG. 1 illustrates that most tissue-specific differential DNA methylation was located at CpG island shores. (a) An example of a T-DMR located at a CpG island shore in the PRTFDC1 gene. The upper panel is a plot of M value versus genomic location for brain (gray), liver (pink) and spleen (purple). Each point represents the methylation level of an individual sample for a given probe. The curve represents averaged smoothed M values, described in detail in the Methods. Because of the scale and standardization used, M values that range from −0.5 to 0.5 represented unmethylated sites as defined by the control probes, and values from 0.5 to 1.5 represent baseline levels of methylation. The middle panel provides the location of CpG dinucleotides with black tick marks on the x axis. CpG density was calculated across the region using a standard density estimator and is represented by the smoothed black line. The location of the CpG island was denoted on the x axis as an orange line. The lower panel provides gene annotation for the genomic region. The thin outer gray line represents the transcript, the thin inner lines represent a coding region. Filled in gray boxes represent exons. On the y axis, plus and minus marks denote sense and antisense gene transcription, respectively. (b) An example of a C-DMR that was located in a CpG island shore and that overlapped a T-DMR. Liver (pink) was hypomethylated relative to brain (gray) and spleen (purple) tissues. Hypomethylation of colon tumor (orange) was observed in comparison to matched normal colon tissue (green) and overlapped the region of liver hypomethylation.

FIG. 9 illustrates that genome-wide DNAm analysis showed methylation variation was more common outside of CpG islands than within them. The average M value was computed for four sets of normal brain, liver, spleen and colon. Methylation variation across tissues was determined using the standard deviation across tissues of within tissue-averaged M. For all CHARM probes located within a CGI, left plot, and all CHARM probes located outside of a CGI, right plot, a smooth scatter plot was used to represent distribution of values from high (blue) to low (yellow) number of points. Where tissue M variation was greater than 0.75 yellow points were used, and where greater than 1.0, orange points.

Similar CpG island shore hypo- and hypermethylation in cancer. The same comprehensive genome-wide was used approach to address cancer-specific DNA methylation. The focus was on colorectal cancer, a paradigm for cancer epigenetics because of the availability of subject-matched normal mucosa, the cell type from which the tumors arise. DNAm was analyzed on 13 colon cancers and matched normal mucosa from the same individuals, identifying 2,707 regions showing differential methylation in cancers (C-DMRs) with an FDR of 5% (Table 11 describes some of the identified C-DMR regions. The complete set of C-DMRs is available on the Nature Genetics website (nature.com/naturegenetics) see “Supplementary Data 2” in the Supplementary Information for Irizarry et al., Nature Genetics 41(2):178-186). Plots similar to those in FIG. 1 for 10 of the C-DMRs are provided in FIGS. 12A-J; the complete set of C-DMRs plots, ordered by statistical significance, are available online at rafalab.jhsph.edu/c-dmr-all.pdf). These C-DMRs were similarly divided between those showing hypomethylation in the cancer (compared to the normal colon) and those showing hypermethylation (1,199 (44%) and 1,508 (56%), respectively). The CHARM arrays, like other tiling arrays, do not contain repetitive sequences, so the abundance of hypomethylation was not due to enrichment for repetitive DNA, which has been shown to be hypomethylated in cancer. This similarity in amount of hypomethylation and hypermethylation was also shown in a quantile-quantile plot, in which quantiles for the observed average difference between tumor and normal sample Ms are plotted against quantiles from a null distribution constructed with the control (M=0) regions (FIG. 3A).

Although both hypomethylation and hypermethylation in cancer involved CpG island shores, there were subtle differences in the precise regions that were altered. The hypermethylation extended to include portions of the associated CpG islands in 24% of cases (termed ‘overlap’ in FIG. 2), which could account for the island hypermethylation frequently reported in cancer, even though that is not the predominant site of modification. In contrast, the hypomethylation extended to between 2 and 3 kb from the associated island in 10% of cases and was not associated with an island in 35% of cases (FIG. 2).

To confirm differential methylation in colon tumors, additional bisulfite pyrosequencing validation of nine C-DMRs, including five regions showing hypermethylation and four regions with hypomethylation, in an average of 50 primary cancer and normal mucosal samples per gene was carried out. For all of the genes, the pyrosequencing data matched the CHARM data (P values ranging from 10⁻⁴ to 10⁻¹⁷) (FIGS. 3 b-j and Table 3). Thus, CHARM was precise in identifying both T-DMRs and C-DMRs.

Our screening process was effective at identifying known targets of altered DNAm in cancer. For example, 10 of the 25 most statistically significant C-DMRs have previously been reported to show altered DNAm in cancer, for example, WNK2, hypermethylated in glioblastoma (Hong, et al., Proc. Natl. Acad. Sci. USA 104:10974-10979, 2007) and HOXA6, hypermethylated in lymphoid malignancies (Strathdee et al., Clin. Cancer Res. 13, 5048-5055, 2007). However, hundreds of genes not previously described were also identified by this screening. For example, for hypermethylation, we identified genes encoding GATA-2, an important regulator of hematopoetic differentiation (Cantor et al., J. Exp. Med. 205, 611-624, 2008), and RARRES2, whose expression is decreased in intestinal adenomas (Segditsas et al., Hum. Mol. Genet. 17:3864-3875, 2008). For hypomethylation, we identified genes encoding DPP6, a biomarker for melanoma (Jaeger et al., Clin. Cancer Res. 13, 806-815, 2007), MRPL36, a DNA helicase that confers susceptibility to breast cancer (Seal et al., Nat. Genet. 38, 1239-1241, 2006), and MEST, a known target of hypomethylation and loss of imprinting in breast cancer (Pedersen et al., Cancer Res. 59:5449-5451, 1999). Note that although previous T-DMR screens have focused on CpG islands, which we show account for only 8% of T-DMRs, our screen did identify CpG island loci validated by others as well, for example, PAX6, OSR1 and HOXC12. Thus, cancer, like normal tissues, involves changes in DNAm in CpG island shores, with comparable amounts of hypomethylation and hypermethylation but with subtle differences in the precise distribution of these alterations with respect to the associated CpG island. These differences will have important functional implications for gene expression, as discussed later.

FIG. 2 illustrates the distribution of distance of T-DMRs and C-DMRs from CpG islands. In the plots, bars denoted “Islands” were regions that cover or overlap more than 50% of a CpG island. Bars denoted “Overlap” were regions that overlap 0.1-50% of a CpG island. Regions denoted by (0, 500) did not overlap islands but are located <500 by of islands. Regions denoted by (500, 1,000) were located >500 and ≦1,000 by from an island. Regions denoted by (1,000, 2,000) were regions >1,000 by and ≦2,000 by from an island. Regions denoted by (2000, 3000) were located >2,000 by and ≦3,000 by from an island. Regions denoted >3,000 were >3,000 by from an island. The percentage of each class was provided for CpG regions (the CHARM arrays themselves, null hypothesis), tissue-specific differentially methylated regions (T-DMRs), cancer-specific differentially methylated regions (C-DMRs), and the latter subdivided into regions of cancer-specific hypermethylation and hypomethylation.

FIG. 3 illustrates similar numbers of sites of hypomethylation and hypermethylation in colon cancer. (a) A quantile-quantile plot showed a similar number of sites of hypomethylation and hypermethylation in colon cancer. The quantiles of the differences in M values between tumor and normal colon tissues were plotted against the quantiles of a null distribution formed using the differences seen in the control regions. Points deviating from the diagonal were not expected by chance, and a similar proportion is seen for hypomethylation and hypermethylation in cancer. (b-j) Bisulfite pyrosequencing confirms the prevalence of five hypermethylated and four hypomethylated C-DMR shores in a large set of colon tumor and normal mucosa samples. Box plots represent degree of DNA methylation as measured using bisulfite pyrosequencing. (b) DLX5 (distal-less homeobox 5). (c) LRFN5 (leucine-rich repeat and fibronectin type III domain containing 5). (d) HOXA3 (homeobox A3). (e) SLITRK1 (SLIT and NTRK like family, member 1). (f) FEZF2 (FEZ family zinc finger 2). (g) TMEM14A (transmembrane protein 14A). (h) ERICH1 (glutamate-rich 1). (i) FAM70B (family with sequence similarity 70, member B). (j) PMEPA1 (prostate transmembrane protein, androgen induced 1). n, number of samples analyzed by pyrosequencing.

Gene expression is linked to non-CpG-island methylation. Because the identification of CpG island shores was unexpected, the functional relationship between their differential methylation and the expression of associated genes was explored. To address tissue- and cancer-specific DNAm, gene expression was analyzed across the genome in five primary brains and livers from the same autopsy specimens. and in four colon cancers and subject-matched normal mucosa; all samples were from subjects for whom genome-wide methylation analysis data had been collected. Methylation of T-DMRs showed a strong inverse relationship with differential gene expression, even though these DMRs were not CpG islands but rather CpG island shores. The relationship between DNAm and gene expression was greater for DMRs in which one of the two measured points had approximately no methylation (‘none-to-some’ methylation compared to ‘some-to-more’ or ‘some-to-less’ methylation), particularly for hypomethylation (FIG. 4). The significant association of gene expression with T-DMRs was true even when the DMR was 300-2,000 by from the transcription start site, for example, average log-ratio values of 0.84 and 0.35 (P<10⁻³⁷ and 10⁻⁴) for some-to-none and some-to-more/less methylation, respectively, comparing liver to brain expression (FIG. 4). Moreover, when T-DMRs were related to changes in gene expression from over 242 samples, representing 20 different tissue types, it was found that 5,352 of the 8,910 genes that were differentially expressed across the 20 tissues were within 2 kb of a T-DMR, much more than expected by chance (P<10⁻¹⁵). For C-DMRs as well, even though there were fewer of them than T-DMRs, there was a significant association of gene expression with DNAm: P<10⁻⁶ and P<10⁻³ for hypermethylation and hypomethylation, respectively; again, the relation was much more marked when one of the two measured points had no methylation (FIGS. 10A-J; the complete set is available on the Nature Genetics website (nature.com/naturegenetics) see “Supplementary FIG. 2” in the Supplementary Information for Irizarry et al., Nature Genetics 41(2):178-186).

The inverse relationship between DNAm and transcription was validated at eight CpG island shores, two T-DMRs and six C-DMRs in tissues and colon cancers, respectively, using quantitative real-time PCR. Both of the T-DMRs were in shores, one located 844 by upstream of the promoter and one within the gene body. Similarly, all six of the C-DMRs assayed were in shores, with five located in the gene promoter and one within the gene body (Table 4). These quantitative data provided additional support for a strong relationship between differential methylation in CpG island shores and transcription of associated genes. This functional relationship between gene expression and shore methylation applies to shores located within 2 kb of an annotated transcriptional start site but leaves open the possibility of additional regulatory function for shores located in intragenic regions or gene deserts.

FIG. 4 shows that gene expression was strongly correlated with T-DMRs at CpG island shores. For each brain versus liver T-DMR, the closest annotated gene on the Affymetrix HGU133A microarray was found, resulting in a total of 2,041 gene-T-DMR pairs. Plotted are log(base 2) ratios of liver to brain expression against ΔM values for liver and brain DNAm. Orange dots represented T-DMRs located within 300 by from the corresponding gene's transcriptional start site (TSS). Green dots represented T-DMRs that were located from 300 to 2,000 by from the TSS of an annotated gene. Black dots, in the middle, represented log ratios for all genes further than 2 kb from an annotated TSS.

FIG. 10 illustrates that most tissue-specific differential DNA methylation were located at CpG island shores. The top 50 T-DMRs, ordered by statistical significance. Displays are as in FIG. 1 a. The upper panels are plots of M value versus genomic location for brain (grey), liver (pink), and spleen (purple). Each point represents the methylation level of an individual sample for a given probe. The curve represents averaged smoothed M values, described in detail in the Methods. Due to the scale and standardization used, M values which range from −0.5 to 0.5 represented unmethylated sites as defined by the control probes, and values from 0.5 to 1.5 represented baseline levels of methylation. The middle panels provide the location of CpG dinucleotides with black tick marks on the xaxis. CpG density was calculated across the region using a standard density estimator and is represented by the smoothed black line. The location of the CpG island was denoted on the x-axis as an orange line. The lower panels provide gene annotation for the genomic region. The thin outer grey line represents the transcript, the thin inner lines represent a coding region. Filled in grey boxes represent exons. On the y-axis, plus and minus marks denote sense and antisense gene transcription respectively.

Shore-linked silencing reversed by methyltransferase inhibition. The previous data, although compelling, are associative in nature. For a more functional analysis, DNA methylation and gene expression data from tissues studied in the current work were compared to a rigorous analysis using hundreds of expression microarray experiments published earlier (Gius et al., Cancer Cell 6:361-371, 2004), which tested the effects on gene expression of 5-aza-2′-deoxycytidine (AZA), and also to double DNA methyltransferase 1 and 3B somatic cell knockout (DKO) experiments. Genes from the present study that had DMRs meeting an FDR <0.05 and that showed differential expression in the tissues at P<0.05 were compared to genes that had significant P values after AZA or DKO. Of 27 DMRs that showed relative hypermethylation with gene silencing in tissues, 23 were activated by AZA (FIG. 5 a and Tables 12 and 13). Similarly, of 25 DMRs that showed relative hypermethylation with gene silencing in tissues, all 25 were activated by DKO (FIG. 5 b and Tables 12 and 13). Thus, both chemical and genetic demethylation cause changes in gene expression similar to those associated with increased methylation of CpG island shores.

FIG. 5 shows that genes downregulated in association with T-DMR shore hypermethylation were activated by 5-aza-2′-deoxycytidine treatment of colon cancer cell line HCT116 and knockout of DNA methyltrasferase 1 and 3b in HCT116. (a) Genes significantly upregulated (P<0.05) after treatment of HCT116 cells with 5-aza-2′-deoxycytidine (AZA) (black) that were also associated with a relatively hypermethylated T-DMR showing a significant change in gene expression (P<0.05) (gray): 23/27 genes are activated by AZA. (b) Genes significantly upregulated (P<0.05) after knockout of DNA methyltransferases 1 and 3b (DKO) in HCT116 cells (black) that were also associated with a relatively hypermethylated T-DMRs showing a significant change in gene expression (P<0.05) (gray): 25/25 genes were activated by DKO. Plotted are log(base 2) ratios of expression of AZA/untreated, DKO/HCT116 and relatively hypermethylated/hypomethylated tissue.

DMRs are associated with alternative transcription. The question as to what the function of differential methylation at CpG island shores might be was next addressed. One possibility was alternative transcription. Both the T-DMRs and C-DMRs often involved alternative transcripts, as defined by cap analysis gene expression (CAGE): 68% and 70% of the T-DMRs and C-DMRs, respectively, were not within 500 by of an annotated transcriptional start site but were within 500 by of an alternative transcriptional start site. By chance, only 58% were expect to have this relationship (P<10⁻¹⁵). These results suggested that DNA methylation might regulate alternative transcription in normal differentiation and cancer. Rapid amplification of cDNA ends (RACE) experiments were therefore carried out in order to confirm the presence of alternative transcripts and their differential expression in cancer. Three colon tumor and subject-matched normal mucosa were examined at the PIP5K1A locus, a C-DMR that is hypomethylated in colon tumors, and confirmed that an alternative RNA transcript is produced in colon tumors compared to their matched normal counterparts (B online). Thus, a key function for differential methylation during differentiation may be alternative transcription, and the role of altered DNAm in cancer may in part be disruption of the regulatory control of specific promoter usage.

FIG. 11 illustrates that bisulfite pyrosequencing confirmed the prevalence of 4 hypomethylated C-DMR shores in a large set of colon tumor and normal mucosa samples. Box-plots represent DNA methylation level measured using bisulfite pyrosequencing. a, transmembrane protein 14A (TMEM14A); b, glutamate-rich 1 (ERICH1); c, family with sequence similarity 70, member B (FAM70C); d, prostate transmembrane protein, androgen induced 1 (TMEPA1), (n) equals the number of samples analyzed by pyrosequencing.

Mouse DNAm discriminates human tissues, even far from genes. A compelling argument for the functional importance of differential DNAm of CpG island shores would be their conservation across species. One might expect DMRs near transcriptional start sites to be conserved because the genes are conserved. However, when the relationship between gene-distant T-DMRs (2-10 kb away from an annotated gene) and sequence conservation using the phastCons28way table from the University of California Santa Cruz genome browser was examined, it was found that 48% of differentially methylated regions showed sequence conservation. Furthermore, 91% of DMRs were located within 1 kb of a highly conserved region (P<0.001).

To address whether the DNA methylation itself is conserved across species, a mouse CHARM array was created with ˜2.1 million features independently of the human array. Tissue replicates were then isolated from each of three mice, corresponding to the tissues examined in the human T-DMR experiments, and then mapped these methylation data across species using the UCSC LiftOver tool. The interspecies correspondence of tissue-specific methylation was notable, and unsupervised clustering perfectly discriminated among the tissues, regardless of the species of origin (FIG. 6; P<10⁻⁹). Perfect discrimination among the tissues was found even when the analysis was limited to gene-distant DMRs (FIGS. 12A-J; the complete set is available on the Nature Genetics website (nature.com/naturegenetics) see “Supplementary FIG. 4” in the Supplementary Information for Irizarry et al., Nature Genetics 41(2):178-186)). Thus, DNAm itself is highly conserved across 50 Myr of evolution (approximately 51% of mapped DNAm sites were conserved). It was also noticed relatively little heterogeneity in tissue-specific methylation in the mouse compared to the human (height of the cluster bars in FIG. 6), suggesting a genetic-epigenetic relationship, as the mice are inbred.

FIG. 6 shows clustering of human tissue samples using mouse T-DMRs resulted in perfect discrimination of tissues. The M values of all tissues from the 1,963 regions corresponding to mouse T-DMRs that mapped to the human genome were used for unsupervised hierarchical clustering. By definition, the mouse tissues were segregated. Notably, all of the human tissues were also completely discriminated by the regions that differ in mouse tissues. The three major branches in the dendrograms corresponded perfectly to tissue type regardless of species. Columns represent individual samples, and rows represent regions corresponding to mouse T-DMRs. The heat map shows M values, with red being more methylated and blue less.

FIG. 12 illustrates that most cancer-specific differential DNA methylation was located at CpG island shores. The top 50 C-DMRs, ordered by statistical significance. Displays are as in FIG. 1 b. The upper panels are plots of M value versus genomic location for brain (grey), liver (pink), spleen (purple), normal colon (green), and colon cancer (orange). Each point (shown only for normal colon and colon cancer) represented the methylation level of an individual sample for a given probe. The curve represents averaged smoothed M values, described in detail in the Methods. Due to the scale and standardization used, M values which range from −0.5 to 0.5 represent unmethylated sites as defined by the control probes, and values from 0.5 to 1.5 represent baseline levels of methylation. The middle panels provide the location of CpG dinucleotides with black tick marks on the x-axis. CpG density was calculated across the region using a standard density estimator and is represented by the smoothed black line. The location of the CpG island is denoted on the x-axis as an orange line. The lower panels provide gene annotation for the genomic region. The thin outer grey line represents the transcript, the thin inner lines represent a coding region. Filled in grey boxes represent exons. On the y-axis, plus and minus marks denote sense and antisense gene transcription respectively.

The location of C-DMRs overlaps that of T-DMRs. Because both C-DMRs and T-DMRs were located at CpG island shores, we then asked whether they occurred in similar locations. DMRs in which the methylation difference was from no methylation to some methylation, that is, those DMRs for which the gene expression data above showed a strong relationship between ‘none-to-some’ methylation and gene silencing were focused on. Notably, it was found that 52% of the C-DMRs overlapped a T-DMR, compared to only 22% expected by chance (P<10⁻¹⁴), when using an FDR of 5% for defining T-DMRs. Although these data are significant, the definition of a T-DMR based on FDR of 5% is conservative. It was therefore also asked directly whether C-DMRs are enriched for tissue variation in DNAm by computing an averaged F-statistic (comparison of cross-tissue to within-tissue variation) at each C-DMR. The cross-tissue variation in normal tissues was significant at 64% of the C-DMRs, compared to 20% of randomly selected CpG regions on the array matched for size (P<10⁻¹⁴³). When DMRs were defined using an FDR of 5%, 1,229 of 2,707 C-DMRs overlapped a T-DMR, of which 265, 448 and 185 are brain-, liver- and spleen-specific, respectively, and 331 show variation among all of the tissues (Table 14; the complete data set is available on the Nature Genetics website (nature.com/naturegenetics) see “Supplementary Data 4” in the Supplementary Information for Irizarry et al., Nature Genetics 41(2):178-186). The colon C-DMRs were highly enriched for overlap with liver T-DMRs (P<10⁻¹⁵), and liver was embryologically closest to colon of the autopsy tissues studied. For example, the C-DMR located in the CpG island shore upstream of the HS3ST4 (heparan sulfate D-glucosaminyl 3-O-sulfotransferase 4) gene is hypomethylated in colon cancer compared to normal colon and coincides with a T-DMR that distinguishes liver from other tissues (FIG. 1 b). The correspondence between C-DMRs and T-DMRs was so marked that when unsupervised clustering of the normal brain, liver and spleen, using the M values from the C-DMRs was carried out, there was perfect discrimination of the tissues (FIG. 7).

Most tissue-specific methylation difference more commonly involves hypomethylation, although this varies by tissue type with 50% of liver, 62% of spleen, and 79% of brain DMRs representing hypomethylation, and cancer-specific methylation differences slightly more frequently involve hypermethylation (56%:44%). For both T-DMRs and C-DMRs, when there was differential methylation, it was common that at least one of the tissues was completely unmethylated (68% and 37%, respectively). Furthermore, hypomethylated C-DMRs were twice as likely to resemble another tissue type, such as liver, than were hypermethylated C-DMRs (82% versus 61%, P<10⁻³¹), even though hypermethylated C-DMRs overlapped T-DMRs 1.5-fold more frequently than did hypomethylated C-DMRs (54% versus 35%, P<10⁻²¹).

To further explore the relationship between differentiation and type of methylation change, Gene Ontology (GO) analysis was carried out for both hypomethylated and hypermethylated C-DMRs in the cancers (see Methods). The GO analysis showed enrichment for development and pluripotency-associated genes for both hyper- and hypomethylated C-DMRs (P<0.01) (Table 5). Hypomethylated C-DMRs were also enriched for genes associated with differentiated cellular functions for lineages other than the colon (P<0.01) (Table 5). Thus, cancer-specific DNA methylation predominantly involves the same sites that show normal DNAm variation among tissues, particularly at genes associated with development.

Next, the magnitude of differential methylation and variation in C-DMRs and T-DMRs were examined. The ΔM values for tissue and cancer DMRs differed markedly from nonmethylated controls or randomly selected regions (the latter have an average value comparable to controls but with significant tails, as by definition they may contain DMRs themselves) (FIGS. 8 a-d). The ΔM values for normal tissues were comparable across the tissues, but the ΔM values between normal and cancer tissues were on average approximately half the ΔM between normal tissue pairs (FIG. 8 e), which is logical given that the cancers are compared with their tissue of origin. Another difference between cancer and normal tissues was an increase in the inter-individual variation in M among the colon cancers, which was on average ˜50% greater than the inter-individual variation among the normal colons (FIG. 8 f), a result which may help to explain tumor cell heterogeneity. Given the strong inter-individual variability found in cancer, 205/2,707 C-DMRs were identified that were consistently differentially methylated between the colon tumor and matched normal mucosa from all 13 individuals examined (Table 18; the complete data set is available on the Nature Genetics website (nature.com/naturegenetics) see “Supplementary Data 4” in the Supplementary Information for Irizarry et al., Nature Genetics 41(2):178-186). These regions, heavily over-represented for development and morphogenesis genes, provide a smaller, more focused set of regions for biomarker discovery and carcinogenesis studies.

FIG. 7 shows clustering of normal tissue samples using C-DMRs resulted in perfect discrimination of tissues. The M values of all tissues from the 2,707 regions corresponding to C-DMRs were used for unsupervised hierarchical clustering. (a) By definition, the colon tumors and matched normal mucosa were segregated. The two major branches in the dendrograms corresponded perfectly to tissue type. (b) Notably, all of the normal brains, spleens and livers were also completely discriminated by the regions that differ in colon cancer. The three major branches in the dendrograms corresponded perfectly to tissue type. Columns represent individual samples, and rows represent regions corresponding to C-DMRs. The heat map shows M values, with red being more methylated and blue less.

FIG. 8 shows the magnitude of differential methylation and variation in C-DMRs and T-DMRs. (a-d) Box plots of average ΔM values over all DMRs, compared to randomly chosen regions and unmethylated control regions, matched for length. (a) Liver versus brain. (b) Spleen versus brain. (c) Spleen versus liver. (d) Colon cancer versus normal colonic mucosa. (e) Differences in DNA methylation were greater in magnitude among normal tissues than were differences between colon tumors and matched normal mucosa. For all DMRs the average ΔM was computed. These values were then stratified into T-DMRs, hypermethylated C-DMRs and hypomethylated C-DMRs. T-DMRs were further stratified according to brain versus liver, brain versus spleen and liver versus spleen pairwise comparisons. The box plots represented absolute values of the ΔMs. (f) Inter-individual variation in M was larger among colon tumors than matched normal mucosa. For each C-DMR the average interindividual s.d. of the M values was computed. The box plots represent these values for normal colon mucosa and colon tumors.

FIG. 15 illustrates that gene expression was strongly correlated with C-DMRs at CpG island shores. For each colon tumor versus normal mucosa C-DMR the closest annotated gene on the Affymetrix HGU133A microarray was found, resulting in a total of 650 gene/C-DMR pairs. Plotted are log(base 2) ratios of colon tumor to normal expression against delta M values for colon tumor and normal DNAm. Orange dots represent C-DMRs located within 300 by from the corresponding gene's transcriptional start site (TSS). Green dots represent C-DMRs that are located from 300-2000 by from the TSS of an annotated gene. Black dots, in the middle, represent log ratios for all genes further than 2 kb from an annotated TSS.

FIG. 16 illustrates that differences in DNA methylation were greater in magnitude among normal tissues than were differences between colon tumors and matched normal mucosa. For all DMRs the average delta M was computed. These values were then stratified into T-DMRs, hypermethylated C-DMRs and hypomethylated C-DMRs. T-DMRs were further stratified according to brain versus liver, brain versus spleen, and liver versus spleen pair-wise comparisons. The box-plots represent absolute values of the delta Ms.

FIG. 17 shows that inter-individual variation in M was larger among colon tumors than matched normal mucosa. For each C-DMR the average inter-individual standard deviation of the M-values were computed. The box-plots represent these values for normal colon mucosa and colon tumors.

FIG. 18 demonstrates alternate gene transcription origin in colon tumors at a CpG island shore. A schematic overview of the PIP5K1A gene locus. C-DMR represents the genomic region that is hypomethylated in colon tumors as compared to normals. b, the arrow indicates a 220 bp fragment present in 3 colon tumors but not normal mucosa from the same individual amplified by the Sp3 primer and the 5′ RACE anchor primer.

FIG. 19 shows an example of non-conventional CpG island, a C-DMR that was not adjacent to a conventionally defined CpG island. Upper panel: plot of M value versus genomic location for brain (grey), liver (pink), spleen (purple), normal colon (green), colon tumor (orange). Each point represented the methylation level of an individual sample for each probe across the region. The smoothed line represents averaged, smoothed M values for each tissue, described in detail in the methods. M values which range from −0.5 to 0.5 represent unmethylated points and values from 0.5 to 1.5 represent baseline levels of methylation due to the scale and standardization used. The middle panel provides the location of CpG dinucleotides with black tick marks on the x-axis. CpG density was calculated across this region using a standard density estimator and is represented by the smoothed black line. The location of non-conventional CpG islands, defined using Hidden Markov modeling, is denoted on the x-axis as an orange line. The lower panel provides gene annotation for the genomic region. The thin outer grey line represents the start of transcription, the thin inner lines represent the start and end of a coding region. Filled in grey boxes represent exons. On the y-axis, plus and minus marks denote sense and antisense gene transcription respectively.

FIG. 20 illustrates the methylation of mouse T-DMRs completely discriminated tissues regardless of species of origin, even for T-DMRs >2 kb from an annotated gene. The M values of all tissues from the 957 regions corresponding to mouse T-DMRs that mapped to the human genome and were greater than 2 kb from an annotated gene were used for unsupervised hierarchical clustering. By definition, the mouse tissues were segregated. Surprisingly, all of the human tissues were also completely discriminated by the regions that differ in mouse tissues. The three major branches in the dendrograms correspond perfectly to tissue type regardless of species. Columns represent individual samples, and rows represent regions corresponding to mouse T-DMRs. The heatmap displays M values, with red being more methylated and blue less.

Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

TABLE 1 Bisulfite pyrosequencing confirms differential DNA methylation at CpG island shores but not the associated island. Gene Location^(a) Region Tissue^(b) CG1 CG2 CG3 CG4 CG5 CG6 CG7 CG8 CG9 PCDH9 +3,338 Shore Brain 32 26 12 39 19 22 Spleen 91 71 31 76 66 60 P value <.001 <.001 <.001 <.001 <.001 0.003 −267 Island Brain 2 3 4 2 3 5 2 3 2 Spleen 2 3 3 2 3 6 2 4 3 P value 0.032 0.298 0.336 0.108 0.475 0.150 0.393 0.141 0.011 HEY1 +3,381 Shore Brain 54 53 51 51 Liver 70 84 87 71 P value .023 <.001 <.001 <.007 +2,207 Island Brain 4 7 3 4 4 5 1 8 5 Liver 3 6 3 4 4 6 2 9 23 P value 0.349 0.309 0.226 0.460 0.630 0.252 0.017 0.336 0.255 HAGH +2,192 Shore Liver 26 30 22 18 7 6 23 33 Spleen 93 93 82 56 20 20 86 95 P value <.001 <.001 <.001 <.001 <.001 <.001 0.017 0.017 +206 Island Liver 2.1 1.1 2.1 3.4 2.3 6.7 2.7 2.2 3 Spleen 2.2 1.6 2.9 3.7 2.2 2.2 2.1 2.1 3.6 P value 0.608 0.207 0.433 0.803 0.058 0.342 0.262 0.529 0.504 SLMO2 +1,125 Shore Normal 89 63 85 46 68 30 78 81 75 Tumor 37 28 34 19 30 13 34 40 35 P value <.001 <.001 <.001 0.005 0.002 <.001 <.001 <.001 0.002 +40 Island Normal 4 2 3 3 6 4 3 2 3 Tumor 4 1 3 3 3 4 3 2 2 P value 0.619 0.233 0.293 0.546 0.302 0.364 0.461 0.204 0.586 Gene Location^(a) Region Tissue^(b) CG10 CG11 CG12 CG13 CG14 CG15 CG16 CG17 CG18 PCDH9 +3,338 Shore Brain Spleen P value −267 Island Brain 3 3 3 5 5 4 3 3 4 Spleen 3 3 3 3 5 4 3 4 3 P value 0.661 0.265 0.208 0.420 0.051 0.133 0.885 0.783 0.270 HEY1 +3,381 Shore Brain Liver P value +2,207 Island Brain 5 7 7 4 Liver 26 26 8 8 P value 0.179 0.238 0.432 0.001 HAGH +2,192 Shore Liver Spleen P value +206 Island Liver 1.1 2.2 1.8 1.2 1.4 0.6 1.7 0.6 4.2 Spleen 1.2 4.4 1.9 1.8 2.5 2.1 4.4 0.8 7.8 P value 0.782 0.060 0.832 0.366 0.074 0.307 0.073 0.141 0.015 SLMO2 +1,125 Shore Normal 82 40 85 81 43 65 76 76 87 Tumor 36 18 38 36 19 30 39 37 46 P value <.001 0.036 <.001 <.001 <.001 <.001 <.001 0.003 <.001 +40 Island Normal 2 3 3 4 2 7 5 Tumor 4 3 2 4 2 7 6 P value 0.263 0.173 0.369 0.253 0.928 0.230 0.509 ^(a)Location represents distance in base pairs, + denoting upstream and − downstream, from the transcriptional start site to the closest CpG site measured by bisulfite pyrosequencing (CG1). CG1-18 denote individual CpG site measured by bisulfite pyrosequencing. Values are percent methylation. The coordinates for each CpG site measured by pyrosequencing are provided in Supplementary Table 7. ^(b)Brain, spleen, and liver tissues are from the same individuals. Normal and tumor represent matched colon tumor and mucosa from the same individuals.

TABLE 2 Bisulfite pyrosequencing confirms differential DNA methylation at 4 additional CpG island shores Gene Location^(a) Region Tissue^(b) CG1 CG2 CG3 CG4 CG5 CG6 SEMA3C −409 Shore Tumor 31 32 32 34 31 26 Normal 65 71 68 64 63 52 P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 DSCAML1 +2,875 Shore Brain 34 30 29 28 33 26 Liver 86 79 87 89 76 71 P value <0.001 <0.001 <0.001 <0.001 <0.001 0.004 GPT2 −2,984 Shore Spleen 93 93 82 56 20 20 Liver 26 30 22 18 7 5 Pvalue <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 ZNF532 +1,127 Shore Liver 97 96 92 93 79 96 Brain 34 38 26 30 23 24 Pvalue <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Gene Location^(a) Region Tissue^(b) CG7 CG8 CG9 CG10 CG11 CG12 SEMA3C −409 Shore Tumor 24 21 23 9 9 1 Normal 58 41 43 21 17 2 P value 0.007 <0.001 0.001 0.003 <0.001 0.048 DSCAML1 +2,875 Shore Brain 33 26 38 23 26 Liver 88 82 98 70 77 P value <0.001 <0.001 <0.001 <0.001 <0.001 GPT2 −2,984 Shore Spleen 86 95 Liver 23 33 Pvalue <0.001 <0.001 ZNF532 +1,127 Shore Liver 98 89 Brain 22 21 Pvalue <0.001 <0.001 ^(a)Location is distance in base pairs, + denoting upstream and − downstream, from the transcriptional start site to closest CpG site measured by bisulfite pyrosequencing (CG1). CG1-12 denote individual CpG sites measured by bisulfite pyrosequencing. Values are percent methylation. The coordinates for each site are provided in Supplementary Table 7. ^(b)Brain, spleen, and liver tissues are from the same individuals. Normal and tumor represent matched colon tumor and mucosa from the same individuals.

TABLE 3 Bisulfite pyrosequencing confirms differential DNA methylation at 9 C-DMRs in colon tumor and normal colon mucosa samples. C-DMR Gene CHARM methylation^(a) Normal mean^(b) Tumor mean^(b) CN^(c) CT^(c) Matched^(d) P Chr7: DLX5 T > N 8 22 53 61 42 <10⁻⁶ 96491680-96494485 Chr14: LRFN5 T > N 23 44 61 65 55 <10⁻¹⁴ 41147653-41148722 Chr7: HOXA3 T > N 52 72 52 59 44 <10⁻¹⁶ 27129188-27131713 Chr13: SLITRK1 T > N 36 46 56 51 26    0.0009 83352750-83353823 Chr3: FEZF2 T > N 16 40 42 48 39 <10⁻¹⁴ 62335415-62336514 Chr6: TMEM14A N > T 67 45 57 64 53 <10⁻¹¹ 52637426-52638797 Chr8: ERICH1 N > T 71 56 30 34 23 <10⁻³ 846979-849372 Chr13: FAM70B N > T 67 50 38 36 29 <10⁻³ 113615559-113616275 Chr20: TMEPAI N > T 29 17 48 41 30 <10⁻⁶ 55705356-55707713 CN = normal colon tissue; CT = colon tumor ^(a)Methylation level reported by CHARM from greatest to least. ^(b)Mean methylation level reported by bisulfite pyrosequencing. ^(c)Total number of colon samples included in the bisulfite pyrosequencing mean methylation level reported. ^(d)Total number of colon tumor and normal samples from the same individual reported in the bisulfite pyrosequencing mean methylation level.

TABLE 4 Relationship between DNA methylation and gene expression for 2 T-DMRs and 6 C-DMRs Fold Gene Methylation^(a) Region Type Distance^(b) Distance^(c) change^(d) P Expression^(e) FZD3 Brain < Liver Upstream T-DMR 844 bp 7 bp 28.2 0.002 Brain > Liver Brain < Spleen Shore 844 bp 7 bp 34.0 <0.001 Brain > Spleen RBM38 Brain > Spleen Intragenic T-DMR 1937 bp  69 bp  7.0 0.005 Brain < Spleen Liver > Spleen Shore 1937 bp  69 bp  4.0 0.136 Liver < Spleen NDN Tumor > Normal Promoter C-DMR  47 bp 0 bp 4.2 0.025 Normal > Tumor Shore TRAF1 Tumor > Normal Intragenic C-DMR 724 bp 0 bp 2.8 0.025 Normal > Tumor Shore ZNF804A Tumor > Normal Promoter C-DMR 105 bp 185 bp  2.5 0.047 Normal > Tumor Shore CHRM2 Tumor > Normal Promoter C-DMR 433 bp 0 bp 2.0 0.346 Normal > Tumor Shore NQO1 Normal > Tumor Promoter C-DMR 146 bp 216 bp  2.8 0.004 Tumor > Normal Shore SEMA3C Normal > Tumor Promoter C-DMR 143 bp 0 bp 4.8 0.025 Tumor > Normal Shore ^(a)Methylation level reported by CHARM from greatest to least. ^(b)Base pairs to canonical transcriptional start site from the DMR. ^(c)Base pairs to an alternative transcriptional start site from the DMR. ^(d)Fold change = 2^(−ΔΔCT; ΔΔC) _(T) is equal to (C_(T tissue A target gene −) C_(T beta actin)) − (C_(T tissue B target gene −) C_(T beta actin)). ^(e)Tissue expression from greatest to least. P was computed using a paired, two-tailed, t-test.

TABLE 5 Distribution of C-DMRs and T-DMRs. Total number Percent Cancer DMRs 2,707 100% Colon DMRs Tumor hypermethylated 1,508 56% Tumor hypomethylated 1,199 44% Tissue DMRs 16,379 100% Brain DMRs 8200 Brain hypermethylated 1717 21% Brain hypomethylated 6483 79% Liver DMRs 3511 Liver hypermethylated 1763 50% Liver hypomethylated 1748 50% Spleen DMRs 3186 Spleen hypermethylated 1208 38% Spleen hypomethylated 1978 62% Brain:Liver:Spleen DMRs 1482 Brain:Liver:Spleen DMRs are regions that vary in methylation across all three tissues.

TABLE 6 Anchorage-independent growth of C-DMR associated genes assayed by soft agar analysis. Expression Colonies Colonies Gene Methylation status^(a) Expression^(b) fold change^(c) HeLa HeLa P^(d) HCT116 HCT116 P^(d) NQO1 Hypomethylated T > N 2.8 32 0.01 102 0.069 ZNF804A Hypermethylated N > T −2.5 19 0.27 79 0.005 CHRM2 Hypermethylated N > T −2.0 9 <0.01 81 0.037 Vector N/A N/A N/A 21 N/A 125 N/A ^(a)Methylation level of colon tumor as compared to matched normal mucosa, reported by CHARM. Hypomethylated: some methylation in normal, none in tumor. Hypermethylated: some methylation in tumor, none in normal. ^(b)Tissue expression from greatest to least. ^(c)Fold change = 2^(−ΔΔCT); ΔΔC_(T) is equal to (C_(T tissue A target gene −) C_(T beta actin)) − (C_(T tissue B target gene −) C_(T beta actin)). ^(d)Table shows the number of colonies, n = 4. P was computed using a paired, two-tailed, t-test.

TABLE 7 Location of CpG sites validated by bisulfite pyrosequencing Chromosomal coordinates for CG1-CG9 Gene Region Chr CG1 CG2 CG3 CG4 CG5 SLMO2 Shore 20 57049852 57049859 57049884 57049912 57049933 SLMO2 Island 20 57051256 57051274 57051277 57051280 57051287 PCDH9 Shore 13 66698906 66698964 66699018 66699044 66699123 PCDH9 Island 13 66702731 66702734 66702745 66702747 66702753 HEY1 Shore 8 80839112 80839160 80839206 80839272 HEY1 Island 8 80840367 80840374 80840376 80840382 80840385 HAGH Shore 16 1814967 1814974 1814978 1815044 1815048 HAGH Island 16 1816837 1816840 1816843 1816846 1816848 SEMA3C Shore 7 80387012 80387355 80387019 80387340 80387067 DSCAML1 Shore 11 117170021 117170072 117170149 117170209 117170224 GPT2 Shore 16 45474293 45474335 45474389 45474480 45474502 ZNF532 Shore 18 54683963 54683972 54683997 54684040 54684060 Gene Region CG6 CG7 CG8 CG9 SLMO2 Shore 57049937 57049958 57049972 57050013 SLMO2 Island 57051293 57051299 57051305 57051308 PCDH9 Shore 66699126 PCDH9 Island 66702766 66702770 66702776 66702779 HEY1 Shore HEY1 Island 80840389 80840391 80840399 80840409 HAGH Shore 1815086 1815091 1815095 HAGH Island 1816851 1816868 1816873 1816875 SEMA3C Shore 80387071 80387080 80387092 80387102 DSCAML1 Shore 117170231 117170236 117170239 117170250 GPT2 Shore 45474508 45474539 45474564 ZNF532 Shore 54684099 54684127 54684162 Chromosomal coordinates for CG10-CG18 Gene Region Chr CG10 CG11 CG12 CG13 CG14 CG15 CG16 CG17 CG18 SLMO2 Shore 20 57050041 57050045 57050047 57050057 57050099 57050109 57050116 57050141 57050171 SLMO2 Island 20 57051310 57051317 57051305 57051327 57051329 57051337 57051347 PCDH9 Shore 13 PCDH9 Island 13 66702783 66702789 66702798 66702802 66702816 66702825 66702828 66702838 66702841 HEY1 Shore 8 HEY1 Island 8 80840425 80840435 80840441 80840446 HAGH Shore 16 HAGH Island 16 1816895 1816904 1816909 1816913 1816927 1816929 1816933 1816935 1816953 SEMA3C Shore 7 80387104 80387107 80387120 DSCAML1 Shore 11 117170304 117170311

TABLE 8 Primer sequences and annealing temperatures used for bisulfite pyrosequencing Annealing Nested annealing Gene Region Primer Sequence (5′→3′) temperature (° C.) temperature (° C.) SLMO2 Island Forward ATAATGAGGTATAGAGGTTATA 44 Reverse AACATCTATATCAACAAACTAA Nested forward Bio-GAGGTTATATTTGTTTTTGTTT 45 Nested reverse Bio-AACTCTACCCAAAAATCAAAA Sequencing1 (F) GGTTTTGTTTTAGTTTTG Sequencing2 (R) CAAAACTAAAACAAAACC Shore Forward GATATAGTAGGTTTTAGGATGTGT 49 Reverse TTACCACACTATTTTAATTAATATAACCT Nested forward AGGATGTGTTTTATTGAGTATA 43 Nested reverse Bio-AAAACCATTTATATTTTTAAAACT Sequencing1 (F) TGTTTTATTGAGTATAAATG Sequencing2 (F) GTGGTTTATATTTGTAATTT Sequencing3 (F) GAATTATTTGAGGTTAGGTG Sequencing4 (F) TGATTAATATGGTGAAATTT Sequencing5 (F) TTAAAAATATAAAAATTAGT Sequencing6 (F) GGAGGTTAAGGTAGGAGAAT Sequencing7 (F) GTTGTAGTGAGTTAAGAATA HEY Island Forward GAGGTGATTATAGGGAGTAT 46 Reverse AACCCTAAAATTTTTCTTTTATTC Nested forward GTTTTGGGGTAGTAATAG 41 Nested reverse Bio-CTAAACATCATTAAAAAACTA Sequencing (F) GGTTTTTTAGGGAATGTGTT Shore Forward AAAGAGGTATTATTATTTATATATTTTGTGG 49 Reverse TATTAAACTTAAACCTAAAATTTCACATC Nested forward GGTAGTTTTAGGAAAATTAGG 45 Nested reverse Bio-AACTATTAATAACCCTAAATCC Sequencing1 (F) GTATTATTTAATTGATTAYI7 Sequencing2 (F) ATATTTGTGAATTTGAGATT Sequencing3 (F) TTGGGGTTGGTAAATGTAGG Sequencing4 (F) AATGAGATTTAATTTATTAG HAGH Island Forward TAGGTTTGGTTTTGTTTATTTAG 46 Reverse CAATAACCTAAATACTACCATAATT Nested forward Bio-GTTGTTTAGGATTGTAAAATAT 44 Nested reverse Bio-AAAAAAACCAACTACCTC Sequencing1 (F) TTGTTTTTAGTTAATTAG Sequencing2 (F) GTATAGTGGATTTTTGGAGGT Sequencing3 (R) CTAATTAACTAAAAACAA Sequencing4 (R) ACCTCCAAAAATCCACTATAC Shore Forward AAGGAGTATAATAAGTAGAGTGTG 49 Reverse AAAACACCTCCCTAAATTATCAA Nested forward GTAGAAGGGTTGTGATAGGAT 48 Nested reverse Bio-CCTCCCTAAATTATCAACTTC Sequencing1 (F) GAAGGGTTGTGATAGGATTT Sequencing2 (F) ATAAATAAAAATATTGTTTA Sequencing3 (F) ATTTTAGTATTTTGGGAGGT Sequencing4 (F) AAATATAAAAATTAGTTGGG Sequencing5 (F) GGAGGTTGAGGTAGGAGATT Sequencing6 (F) AGGTAGAGGTTGTAGTGAGT PCDH9 Island Forward GTTGATTGTTTTTTAGTTTTTTT 45 Reverse CGCAACCCAAAAATAACTATA Nested forward Bio-AAATTTGATTTTGGTTTTAGGAGA 49 Nested reverse Bio-ACTCCCCCATCTATACATTTTAA Sequencing1 (F) GTATTGAGTATGTTTTGTAGGGTT Sequencing2 (R) CTTCACTTAACAAAAAAATAT Shore Forward GAAAATGATTATGAGTAAATTTGGG 49 Reverse CTTAAAAATAAAAATAACAACCCACC Nested forward GAGTAAATTTGGGGTTATTGT 48 Nested reverse Bio-CCAATTTTCAACCAACCTATA Sequencing1 (F) AGAATAGTAATAATTATAGT Sequencing2 (F) TAGTTATTGTAAAAATGAAT Sequencing3 (F) TTGTTAAAATTTTTGTTTTT Sequencing4 (F) TAGTTGTTAAGTATAATTTA SEMA3C Shore Forward TGTATTTTTAGTAGAGATAGGGTTAG 49 Reverse TCTATTAACATAACTCAAAACAACC Nested forward TTAGTAGAGATAGGGTTAGG 46 Nested reverse Bio-CAAAACAACCTCTCCACATAA Sequencing1 (F) GATTTTTTGATTTAATGGTT Sequencing2 (F) TTAAAATAGGTTAAGATAAA Sequencing3 (F) TAATTTTGTTGATTTTTTTA Sequencing4 (F) TATTTTTAAATATAATTATA DSCAML1 Shore Forward TAGATATTGAATAGAATGTTGGAGA 49 Reverse ATTATTTCCATTCCTCTCAAAATAC Nested forward AGAATGTTGGAGATTTTTTTAATG 45 Nested reverse Bio-CCTTTTTTTTATAATACCTAAACT Sequencing1 (F) AGATTTTTTTAATGTTTTGA Sequencing2 (F) GAAGAATTATGAGTTTTTAT Sequencing3 (F) GAAAATAGGAATTTTAGTGT Sequencing4 (F) AGGTAATATAGATGTTGGTA Sequencing5 (F) TAAGGTAGTTATTGGAGATT GPT2 Shore Forward ATTGGGGAAGATTTTTATTTAGAG 49 Reverse CTAAATCCCAAATTCTCCATATAC Nested forward AGATTGAATATTTGGTTATTAAG 44 Nested reverse Bio-CTCTAAAATCCTTCCCCTTAA Sequencing1 (F) ATTTAGTGTAGATAAAGGTG Sequencing2 (F) GAGTTTAAATAATTTTTTAG Sequencing3 (F) GGAATATTTAAAGATATTTT Sequencing4 (F) TAGGTGTTATTTTGATTTTA Sequencing5 (F) AAATTTAGTTAAGTAGTGTA ZNF532 Shore Forward TTTAGAGTGGAGAAGAAATGTT 49 Reverse ATATTCCACATTAAACATATACCAC Nested forward GTTTAATGGGATTAGAGTGATTT 46 Nested reverse Bio-ATAAAAAACCTTTCAAATTAACAC Sequencing1 (F) GTGATYFTATGATGGTATTG Sequencing2 (F) GAGTTAGGTTTGGAAGGAAG Sequencing3 (F) ATGTGTTGTTTTGTATAAGA Sequencing4 (F) AAGGAAGGGTTTTATTAAAT DLX5C Shore Forward TATTTAGGGTTATTTGGTTTTTTTT 48 Reverse AACCTAACTCCCTACCCACTTATCT Nested forward Bio-GGATTGTATTAGAAAAATATAGT 43 Nested reverse ACCCATATTTCCCTCCTAT Sequencing (R) CTACAACTCTATTTACCC ERICH1 Shore Forward TATTTTATTGTGGGAGTTTTTGGAG 51 Reverse AATAATCACATTTCTCACTTTTACCACTA Nested forward TGGGAGTTTTTGGAGTATAGT 42 Nested reverse TATATTTACCTATATTCCTATCT Sequencing (R) AATAAAATACATTTATTATCATT FAM70B Island Forward TTTTGTGTTTTGTTGTGGTGTG 52 Reverse CTAACTCCAAACTCCAAAACCATTA Nested forward GGAAATGAGATTTATTGAGAG 46 Nested reverse Bio-CTCTCCTTCTATTACAACTAA Sequencing (F) TAGTTATTGGTAATTTTTAG SLITRK1 Shore Forward TTGATTTTGATTTGTTAGTTGTTTG 48 Reverse TATTCCAATATATACCCATCACCC Nested forward Bio-GGGGTTTAGGAGTAAAGGTT 45 Nested reverse CTACCCTATAAAAAAATCTTAAA Sequencing (R) ATTCCCAAAAATACCCTAAT LRFN5 Shore Forward TTGTTGTGGAGGAGTTTGTTAG 48 Reverse TCCAACCTACTCCTTATAAATC Nested forward TGGTTTGTATGAAAGGGAATAT 49 Nested reverse Bio-CCTACTCCTTATAAATCAAAACACC Sequencing (F) TTTAGTTGTATTGTTTT FEZF2 Shore Forward GTGTGGTTAGAGGTATAAGTAGA 50 Reverse TCAACCCTCTCAAAACTTATTCCTA Nested forward GTAGAGGGAAGAAAAGATTTTTTTT 49 Nested reverse Bio-CCCAATCCTCCCCCTTTC Sequencing (F) TTGTAGATTATTTTATTTG TMEM14A Island Forward TGGGTGGGTGTAGATATTTTGTTAT 54 Reverse ACAATCCTACACACACAAACCTTTA Nested forward Bio-TAGTGAAAGTTTTGGGAAATTTA 48 Nested reverse CTACACACACAAACCTTTAATAA Sequencing (R) ATTCATTTTAAAAAATAATCC TMEPAI Shore Forward TATTAATTAAATTGTTTTTAGGAAGGTAAT 49 Reverse AAATTTACATAAAACCACAACAAAC Nested forward GTTTTTAGGAAGGTAATTAGAA 45 Nested reverse Bio-TACAAAAACTTACCAAATCTATAT Sequencing (F) AAATTTTAAGAAGTTAGTA HOXA3 Island Forward GATTAATGAGTTATAGAGAGATGTTG 48 Reverse AAGGAGTTAAAAGTTTTTGGAG Nested forward GTTTAGGTTTTTTATTTTATAATG 43 Nested reverse TAAGATTTGGTGAGGGTTTGT Sequencing (F) GGGTGATTTATGAA Bio = 5′ biotin added; F = forward; R = reverse

TABLE 9 Real time quantitative RT-PCR assays Gene Assay ID (Applied Biosystems) FZD3 Hs00184043_m1 RBM38 Hs00766686_m1 NDN Hs00267349_s1 TRAF1 Hs00194638_m1 ZNF804A Hs00290118_s1 CHRM2 Hs00265208_s1 NQO1 Hs00168547_m1 SEMA3C Hs00170762_m1

TABLE 10 Regions with tissue-specific differential methylation (T-DMRs) at a FDR of 5%. tissue chr start end fdr Varying brainM liverM spleenM chr1 143786173 143787603 0 brain −0.11535 1.487259 1.302311 chr1 8009359 8011113 0 liver 1.299418 −0.0514 1.426635 chr10 101831054 101831872 0 liver 1.292584 −0.05296 1.304799 chr12 131467604 131472462 0 brain 0.26132 1.456655 1.679197 chr12 6531994 6532981 0 All −0.20912 1.254775 0.874488 chr14 23089148 23090035 0 All −0.63109 1.535749 1.192863 chr14 50629706 50630173 0 All −0.08017 1.408726 1.106452 chr14 59165574 59166710 0 brain −0.14155 1.252699 1.205902 chr15 75709049 75710128 0 brain −0.15474 1.309527 1.226063 chr15 91163167 91164726 0 All −0.47387 1.249181 0.967742 chr16 45472725 45475365 0 liver 1.345268 −0.04038 1.356521 chr16 45477675 45478565 0 All −0.55643 0.63018 1.629881 chr18 54682827 54684488 0 brain −0.46482 1.381573 1.207633 chr2 54538538 54540625 0 All −0.28043 1.191245 0.59945 chr20 60184636 60188417 0 liver 1.335582 −0.15525 1.454903 chr4 99796292 99798038 0 All −0.53255 1.000455 0.477587 chr5 98138988 98139704 0 brain 0.361833 1.754281 1.493639 chr8 17314239 17314991 0 brain −0.11832 1.481885 1.356334 chr8 75425908 75426276 0 All −0.3947 1.254908 0.888546 chrX 144713386 144714243 0 brain −0.03791 1.36192 1.560882 chrX 53131580 53132161 0 brain 0.355145 1.795164 1.815183 chr17 73467744 73468595 1.72E−14 All 0.021405 1.358955 1.058227 chr15 35173578 35174577 1.73E−14 All −0.25443 1.220796 0.644358 chr7 129916095 129916949 2.55E−14 All 0.046312 1.436957 0.996807 chr1 158637654 158638754 2.71E−14 brain −0.28314 1.130432 0.847571 chr11 64877881 64878384 5.10E−14 All 0.041802 1.412251 1.04274 chr6 97479869 97480719 8.58E−14 brain −0.17882 1.149061 1.019161 chr20 6462584 6463175 1.57E−13 brain −0.07902 1.303117 1.070685 chr17 71959149 71959442 1.79E−13 All −0.07652 1.36918 0.89536 chr17 35473766 35475501 2.38E−13 All −0.51389 0.699964 0.36019 chr7 130438711 130440952 2.63E−13 All −0.13901 1.122755 0.781425 chr18 51240890 51242290 3.03E−13 brain −0.29669 1.01559 0.812538 chr14 52687631 52688222 3.07E−13 All −0.40997 0.899323 0.121039 chr19 53707072 53707611 7.25E−13 brain −0.58951 0.757836 0.547046 chr1 242283486 242284310 8.08E−13 brain 0.197052 1.546314 1.564309 chr22 44837327 44840079 9.71E−13 brain −0.02283 1.364865 1.280148 chr22 26520905 26522055 9.74E−13 brain −0.08032 1.409407 1.409504 chr12 111302662 111303654 1.10E−12 brain 0.036707 1.361237 1.307891 chr16 17468105 17468795 1.32E−12 All −0.01385 1.030508 1.451115 chr6 53768814 53769599 1.33E−12 brain 0.134246 1.283679 1.070607 chr12 121944796 121946259 1.42E−12 All −0.55677 0.652273 −0.04003 chr4 76775030 76775602 1.75E−12 brain 0.121772 1.391647 1.250962 chr10 11089463 11090071 2.05E−12 brain 0.0384 1.278444 1.277885 chr19 12974645 12975430 2.81E−12 brain 0.058949 1.312149 1.366242 chr9 137121252 137121872 3.27E−12 brain 0.257197 1.567195 1.364371 chr3 187560575 187561467 3.59E−12 All −0.06283 1.222911 0.556389 chr13 43257042 43257653 4.49E−12 brain 0.003724 1.270532 1.252464 chr14 23083610 23084323 4.84E−12 brain 0.332277 1.600948 1.536168 chr20 4616137 4616745 5.79E−12 All −0.33653 0.970823 0.639435 chr18 72330871 72331443 6.11E−12 All 0.090288 1.251749 0.784134 chr7 75386078 75389998 6.67E−12 liver 1.552091 0.424268 1.543758 chr16 30361878 30364176 7.04E−12 liver 1.264842 −0.02377 1.218326 chr1 234916923 234918037 7.40E−12 brain 0.188523 1.354653 1.349526 chr1 227542393 227543223 8.16E−12 brain 0.301546 1.554823 1.484626 chr9 111443165 111443953 9.23E−12 All −0.30158 0.969521 0.313406 chr21 42514640 42515749 9.51E−12 All 0.229328 1.50122 1.077787 chr21 25934681 25935782 9.57E−12 brain −0.47751 0.721757 0.472824 chr19 58297027 58297933 1.01E−11 All −0.41847 0.779032 0.344519 chrX 48937108 48937860 1.08E−11 brain 0.388972 1.581553 1.611601 chr1 33418284 33418931 1.11E−11 All 0.173928 1.468768 0.907082 chr4 21557703 21559151 1.75E−11 brain 0.093552 1.124707 1.26124 chr17 55509863 55510786 2.04E−11 All −0.59336 0.510877 −0.04011 chr21 45713907 45716550 2.04E−11 liver 0.993849 −0.02669 1.158456 chr2 100303365 100304155 2.09E−11 brain 0.079216 1.422576 1.363524 chr2 9684653 9686112 2.40E−11 brain 0.168387 1.319339 1.180425 chr13 66697473 66700370 2.92E−11 brain −0.11815 1.067169 1.241919 chr8 120290407 120291436 3.22E−11 All 0.464379 −0.30417 1.031852 chr17 63963603 63964386 3.36E−11 brain −0.35063 0.832736 0.572111 chr5 16236252 16237106 3.62E−11 All 1.603336 0.431418 1.063083 chr15 27346324 27347653 3.86E−11 brain 0.177365 1.38589 1.150294 chr8 9795653 9796645 3.97E−11 brain 0.23902 1.519226 1.537175 chr15 21361048 21361947 4.66E−11 brain 0.131271 1.324638 1.362028 chr13 43906353 43906790 4.81E−11 brain 0.212522 1.230147 1.332329 chr20 21536810 21537175 5.08E−11 brain −0.00897 1.355417 1.309454 chr6 87918996 87919649 5.11E−11 All −0.0929 1.110646 0.491515 chr3 161040187 161040531 5.29E−11 All −0.12641 1.043576 0.432139 chr16 85164000 85164607 5.63E−11 spleen 0.046453 0.061563 1.155534 chr17 44038889 44039760 6.03E−11 All 0.112679 0.619602 1.416283 chr12 113601482 113602777 6.22E−11 All −0.20773 0.730495 1.016832 chr22 19463535 19464248 6.27E−11 All 1.134095 0.037807 1.449567 chr9 99783345 99784242 6.35E−11 brain 0.238381 1.423687 1.133009 chr10 104669419 104671001 6.57E−11 brain −0.0864 0.974036 1.172766 chr6 52335546 52337567 6.57E−11 brain −0.14951 0.974944 0.765697 chr8 80837473 80839800 6.74E−11 brain −0.13852 1.134604 0.980399 chr1 200125551 200126951 8.29E−11 All 0.200372 1.343235 1.036733 chr11 117169663 117170865 8.61E−11 brain 0.036012 1.319412 1.355828 chr10 88272523 88272784 8.63E−11 All −0.05095 1.334199 0.977942 chr3 160966443 160967336 9.13E−11 brain 0.193104 1.277767 1.047332 chr10 69992084 69993085 9.34E−11 All −0.03598 1.004592 0.494343 chr10 72314866 72316425 9.55E−11 liver 1.431814 0.293288 1.410689 chr5 17272645 17273667 9.66E−11 All 0.335036 1.530197 1.121364 chr19 57202145 57203221 1.10E−10 brain −0.19286 1.016431 0.97668 chr7 139121047 139122912 1.11E−10 All −0.04716 0.405804 1.070219 chr11 65816795 65817554 1.27E−10 brain 0.047017 1.183867 1.21434 chr12 38783038 38784659 1.35E−10 All 0.13847 0.907052 1.253477 chr18 70271478 70272306 1.48E−10 brain 0.477285 1.616985 1.601359 chr12 8958831 8959862 1.58E−10 All −0.41555 0.731955 0.380007 chr7 35542549 35543379 1.58E−10 liver 1.46152 0.187143 1.513851 chr22 44856437 44856847 1.75E−10 All −0.21002 0.964414 0.570562 chr9 113434267 113436690 1.75E−10 liver 1.110322 −0.06272 1.188455 chr3 182923902 182926235 1.78E−10 All −0.28218 0.719899 0.392404 chr5 87600958 87602603 1.78E−10 brain 0.13372 1.234886 1.288835 chr22 44840824 44842585 2.07E−10 All −0.32082 0.854645 0.468445 chr15 35174959 35175429 2.09E−10 All −0.02782 1.121221 0.668461 chr6 32226447 32227133 2.35E−10 brain −0.12679 1.21501 1.108105 chr6 24468445 24468955 2.49E−10 All 0.204555 1.450305 1.08434 chr11 67912958 67914011 2.65E−10 liver 1.672152 0.496634 1.725968 chr15 49699745 49700848 2.69E−10 All 0.005382 1.100265 0.692709 chr10 79064134 79065116 2.73E−10 brain −0.03703 1.057269 0.936404 chr12 102757515 102758501 2.87E−10 All 0.464408 −0.07212 1.247512 chr1 244954765 244956165 3.36E−10 All 0.94556 0.057658 1.256526 chr9 93748388 93749207 3.38E−10 brain 1.558837 0.457913 0.734517 chr17 69717977 69719999 3.48E−10 brain 0.34338 1.370673 1.257086 chr5 75735889 75737508 3.59E−10 brain 1.018357 −0.06504 0.153059 chr14 72427335 72428501 3.61E−10 All 0.110581 1.172443 0.708188 chr4 108858341 108860158 3.69E−10 All 0.019132 1.121514 0.642438 chr19 12956890 12957636 3.90E−10 brain 0.317379 1.422033 1.317791 chr3 135095342 135095599 4.07E−10 All 0.345701 1.547571 1.201468 chr12 119419408 119420682 4.32E−10 All −0.45368 0.64277 0.357627 chr3 194442270 194443491 4.59E−10 brain 0.454163 1.59038 1.582465 chr18 54678682 54681415 4.71E−10 brain −0.11259 0.889818 0.731549 chr10 105441168 105442058 4.95E−10 All −0.07938 1.057892 0.58793 chr4 41341411 41342111 5.00E−10 brain 0.015783 1.215938 1.249218 chr8 80840243 80842031 5.22E−10 All −0.39056 0.633251 0.035054 chr20 33336159 33337040 5.25E−10 All 0.496151 −0.19458 1.042875 chr1 120056929 120057765 5.64E−10 All 0.268229 −0.0413 1.13111 chr9 102276338 102277457 5.97E−10 brain 0.236111 1.305146 1.117734 chr19 2651477 2652367 6.88E−10 brain −0.00437 1.22873 1.134348 chr11 64858559 64859826 7.01E−10 All −0.13863 0.855243 0.225677 chr12 48766660 48767337 7.56E−10 All 0.48622 1.564671 1.166946 chr14 22839242 22839642 8.19E−10 brain −0.07093 1.088938 0.895761 chr12 54406765 54407517 8.73E−10 All 0.079217 1.142971 0.765836 chr6 71720614 71721830 8.87E−10 All 0.190403 1.277573 0.94305 chr2 113632025 113632600 9.06E−10 liver 1.312823 0.278355 1.091582 chr1 152568347 152569153 1.02E−09 brain 0.382712 1.416759 1.180129 chr19 12986646 12989167 1.02E−09 brain 0.027941 1.171056 0.952401 chr18 53172450 53172776 1.05E−09 All −0.18109 0.986896 0.438065 chr22 42139152 42140849 1.07E−09 brain 0.489183 1.544282 1.580222 chr8 94999343 95000446 1.08E−09 All −0.09493 0.967359 0.532637 chr1 158321798 158322793 1.11E−09 brain 0.259199 1.387606 1.405033 chr10 45328714 45329270 1.14E−09 brain 0.487386 1.627923 1.640054 chr4 113519509 113520087 1.17E−09 brain 0.351665 1.428635 1.42591 chr8 28406277 28406880 1.25E−09 All 0.102019 0.893019 1.44425 chr6 31697055 31698136 1.31E−09 All −0.51951 0.524483 0.169974 chr11 1668090 1669572 1.48E−09 liver 1.099981 0.049418 1.262819 chr9 20609087 20610219 1.53E−09 All −0.38654 0.733972 0.290223 chr4 17390836 17391751 1.57E−09 All −0.01514 0.610289 1.056951 chr7 27144976 27145521 1.67E−09 All 0.104066 0.4646 1.284721 chr13 100100517 100101056 1.69E−09 brain 0.126005 1.270831 1.278393 chr17 67631765 67632456 1.70E−09 brain −0.08216 0.965589 1.168482 chr8 139577318 139577719 1.74E−09 All 0.381754 1.528271 1.20125 chr6 39302949 39304155 1.76E−09 All 1.220726 0.182266 0.696068 chr1 159549832 159551072 1.97E−09 All 0.162422 0.860818 1.165009 chr11 129374246 129376513 2.02E−09 liver 1.442827 0.09828 1.273512 chr6 26332969 26333508 2.10E−09 All 0.091736 1.175461 0.869038 chr10 31359338 31360454 2.14E−09 brain −0.11936 0.893576 0.980227 chr7 36157999 36159166 2.24E−09 liver 1.386701 0.239862 1.31825 chr12 116111015 116111629 2.28E−09 All −0.39965 0.299806 0.840599 chr12 24945492 24947103 2.30E−09 brain 0.018833 1.077256 0.926808 chr19 39954301 39955362 2.35E−09 brain 0.084007 1.085292 0.991353 chr10 124212667 124215516 2.40E−09 brain 0.203019 1.299387 1.287902 chr22 36999241 36999534 2.42E−09 All −0.30703 0.886854 0.445371 chr5 72829082 72829798 2.47E−09 All −0.12689 0.443145 0.969204 chr2 42647983 42648909 2.54E−09 All 0.059632 1.131059 0.652138 chr5 108090358 108091228 2.54E−09 All 0.017378 1.041888 0.707126 chr2 16071365 16072112 2.58E−09 All 0.337041 1.347634 0.952726 chr10 105031159 105031629 2.62E−09 All −0.0494 0.688734 1.016566 chr19 51170609 51171407 2.74E−09 brain 0.264918 1.321571 1.394371 chr4 120029760 120030128 2.89E−09 All −0.27772 0.816238 0.511608 chr13 107664610 107665176 2.98E−09 All −0.05834 1.072254 0.649981 chr1 206056315 206056820 3.15E−09 brain 0.066974 1.098728 1.003981 chr8 26207367 26208013 3.17E−09 brain 0.038493 1.160728 0.947717 chr13 112746460 112748715 3.18E−09 brain 0.427857 1.380589 1.457585 chr2 74585982 74586242 3.20E−09 liver 1.767917 0.640233 1.667118 chr12 7175952 7176212 3.28E−09 All −0.01499 1.1523 0.849905 chr1 109626152 109626919 3.33E−09 brain −0.04827 0.99007 0.876846 chr20 23291758 23292447 3.49E−09 All −0.321 0.800623 0.294726 chr4 166520211 166521230 3.50E−09 brain 0.1225 1.28178 1.22046 chr21 39678541 39679533 3.52E−09 liver 1.326263 0.256805 1.372241 chr5 36278172 36279450 3.64E−09 liver 0.870676 0.007346 1.103939 chrX 53466762 53467199 3.82E−09 All −0.89696 0.218855 −0.22092 chr2 48611532 48612389 3.92E−09 All −0.04989 0.995592 0.480689 chrX 18355897 18356374 4.07E−09 brain −0.3703 0.699427 0.547643 chr19 44581428 44584303 4.17E−09 brain 1.129111 0.159947 0.374427 chr1 165182806 165183330 4.19E−09 All 0.078551 1.160163 0.736041 chr17 68094833 68098785 4.24E−09 brain 0.199103 1.079328 1.153802 chr17 4795197 4796159 4.26E−09 liver 1.18738 0.182585 1.068554 chr19 55744809 55746038 4.26E−09 brain −0.03805 0.758509 1.008509 chr6 2712261 2713113 4.71E−09 All −0.31861 0.757329 0.053413 chr1 207913994 207914866 4.78E−09 All 1.048671 0.009665 0.479916 chr1 228271526 228273514 4.85E−09 All 0.721839 0.263038 1.405718 chr4 3341125 3342144 4.90E−09 brain 0.237939 1.277335 1.144417 chr name annotation region island distTolsla chr1 PDE4DIP NM_022359 overlaps 5′ cover 0 chr1 ERRFI1 NM_018948 upstream Shore 0 chr10 CPN1 NM_001308 overlaps 5′ Far 15878 chr12 GALNT9 NM_021808 upstream Shore 603 chr12 HOM-TES-103 NM_001039670 inside Shore 1705 chr14 THTPA NM_024328 downstream Island 0 chr14 TRIM9 NM_052978 inside Island 0 chr14 RTN1 NM_206852 inside Shore 251 chr15 LRRN6A NM_032808 inside Shore 1495 chr15 CHD2 NM_001042572 downstream Far 9081 chr16 GPT2 NM_133443 downstream Shore 0 chr16 GPT2 NM_133443 inside Shore 1217 chr18 ZNF532 NM_018181 inside Shore 559 chr2 SPTBN1 NM_003128 inside Shore 660 chr20 SS18L1 NM_198935 inside Far 2587 chr4 TSPAN5 NM_005723 inside Shore 127 chr5 RGMB NM_173670 inside Shore 781 chr8 MTMR7 NM_004686 inside Shore 0 chr8 GDAP1 NM_001040875 inside Shore 309 chrX SLITRK2 NM_032539 inside Shore 1945 chrX TSPYL2 NM_022117 inside Far 2034 chr17 FLJ45079 NM_001001685 upstream Shore 197 chr15 MEIS2 NM_172315 inside Shore 101 chr7 MEST NM_177524 inside Shore 1026 chr1 VANGL2 NM_020335 inside Shore 372 chr11 TIGD3 NM_145719 downstream Shore 130 chr6 KIAA1900 NM_052904 inside Shore 487 chr20 BMP2 NM_001200 downstream Far 232877 chr17 UBE2O NM_022066 inside Shore 541 chr17 THRA NM_199334 inside Shore 360 chr7 MKLN1 NM_013255 downstream Shore 0 chr18 TCF4 NM_003199 inside Far 165726 chr14 DDHD1 NM_030637 inside Shore 393 chr19 PSCD2 NM_004228 upstream Shore 320 chr1 ZNF238 NM_006352 inside Shore 587 chr22 C22orf26 NM_018280 upstream Shore 635 chr22 MN1 NM_002430 inside Shore 738 chr12 RPL6 NM_000970 downstream cover 0 chr16 XYLT1 NM_022166 inside Far 869871 chr6 LRRC1 NM_018214 inside Shore 0 chr12 VPS37B NM_024667 inside Shore 27 chr4 CDKL2 NM_003948 upstream Shore 0 chr10 CUGBP2 NM_001025077 inside Far 9377 chr19 NFIX NM_002501 inside Shore 74 chr9 OLFM1 NM_006334 inside Shore 579 chr3 DGKG NM_001346 inside Shore 0 chr13 CCDC122 NM_144974 downstream Shore 205 chr14 THTPA NM_024328 downstream Far 5519 chr20 PRNP NM_000311 inside Shore 263 chr18 FLJ44881 NM_207461 downstream Shore 295 chr7 POR NM_000941 inside Far 3367 chr16 SEPHS2 NM_012248 overlaps 3′ Shore 55 chr1 ACTN2 NM_001103 inside Shore 0 chr1 C1orf96 NM_145257 inside Shore 1414 chr9 PALM2 NM_001037293 inside Shore 0 chr21 ABCG1 NM_207174 inside Shore 1439 chr21 JAM2 NM_021219 inside Shore 412 chr19 ZNF160 NM_033288 inside Shore 0 chrX SYP NM_003179 inside Shore 1759 chr1 TRIM62 NM_018207 inside Shore 269 chr4 KCNIP4 NM_147182 inside Shore 32 chr17 ABC1 NM_022070 inside Shore 6 chr21 COL18A1 NM_030582 inside cover 0 chr2 LONRF2 NM_198461 upstream Shore 56 chr2 YWHAQ NM_006826 inside Shore 1528 chr13 PCDH9 NM_020403 inside Far 2224 chr8 MAL2 NM_052886 inside Shore 4 chr17 WIPI1 NM_017983 inside Shore 558 chr5 FBXL7 NM_012304 upstream Far 2832 chr15 NDNL2 NM_138704 overlaps 3′ Shore 1133 chr8 TNKS NM_003747 upstream Shore 1250 chr15 MKRN3 NM_005664 overlaps 3′ Far 120705 chr13 TSC22D1 NM_006022 inside Far 2361 chr20 NKX2-2 NM_002509 upstream Far 84879 chr6 CGA NM_000735 upstream Shore 0 chr3 SCHIP1 NM_014575 inside Far 74473 chr16 FOXC2 NM_005251 upstream Far 2282 chr17 HOXB7 NM_004502 overlaps 3′ Shore 483 chr12 TBX3 NM_005996 inside Far 2380 chr22 SERPIND1 NM_000185 inside Far 74742 chr9 ANP32B NM_006401 downstream Shore 561 chr10 CNNM2 NM_199077 inside Shore 117 chr6 PAQR8 NM_133367 inside Shore 0 chr8 HEY1 NM_012258 overlaps 3′ Shore 336 chr1 TMEM58 NM_198149 inside Far 60230 chr11 DSCAML1 NM_020693 inside Shore 979 chr10 WAPAL NM_015045 upstream Shore 454 chr3 SCHIP1 NM_014575 inside Shore 729 chr10 CXXC6 NM_030625 inside Shore 1256 chr10 PCBD1 NM_000281 inside Shore 1319 chr5 BASP1 NM_006317 inside Shore 624 chr19 ZNF615 NM_198480 inside Island 0 chr7 TBXAS1 NM_030984 downstream Shore 963 chr11 TMEM151 NM_153266 inside Shore 284 chr12 SLC2A13 NM_052885 inside Shore 570 chr18 C18orf51 NM_001044369 inside Far 2280 chr12 PHC1 NM_004426 inside Shore 84 chr7 HERPUD2 NM_022373 downstream Shore 197 chr22 FLJ27365 NM_207477 downstream Far 3632 chr9 bA16L21.2.1 NM_001015882 inside Shore 241 chr3 SOX2 NM_003106 upstream Shore 865 chr5 TMEM161B NM_153354 upstream Shore 321 chr22 C22orf26 NM_018280 upstream Far 2055 chr15 MEIS2 NM_172315 inside Shore 53 chr6 PRRT1 NM_030651 inside Shore 0 chr6 KAAG1 NM_181337 upstream Shore 55 chr11 LRP5 NM_002335 inside Shore 624 chr15 DMXL2 NM_015263 inside Shore 611 chr10 KCNMA1 NM_002247 inside Shore 985 chr12 NT5DC3 NM_001031701 inside Shore 0 chr1 SCCPDH NM_016002 inside Shore 114 chr9 ROR2 NM_004560 inside Shore 1731 chr17 RPL38 NM_000999 upstream Shore 70 chr5 IQGAP2 NM_006633 inside Shore 0 chr14 DPF3 NM_012074 inside Shore 41 chr4 PAPSS1 NM_005443 inside Shore 136 chr19 NFIX NM_002501 downstream Far 10181 chr3 RAB6B NM_016577 inside Shore 654 chr12 DYNLL1 NM_001037495 overlaps 5′ Shore 312 chr3 HRASLS NM_020386 inside Shore 206 chr18 ZNF532 NM_018181 overlaps 3′ Shore 0 chr10 SH3PXD2A NM_014631 inside Shore 270 chr4 DKFZP686A01247 NM_014988 inside Shore 72 chr8 HEY1 NM_012258 inside Island 0 chr20 FAM83C NM_178468 overlaps 3′ Shore 6 chr1 PHGDH NM_006623 inside Shore 0 chr9 TMEFF1 NM_003692 inside Shore 0 chr19 GNG7 NM_052847 inside Shore 1165 chr11 DPF2 NM_006268 inside Shore 140 chr12 SMARCD1 NM_003076 inside Shore 1064 chr14 BCL2L2 NM_004050 downstream Shore 710 chr12 CD63 NM_001780 inside Shore 767 chr6 B3GAT2 NM_080742 inside Shore 251 chr2 PSD4 NM_012455 downstream Shore 158 chr1 ATP8B2 NM_001005855 inside Shore 195 chr19 NFIX NM_002501 inside Shore 387 chr18 ST8SIA3 NM_015879 inside Shore 0 chr22 MPPED1 NM_001044370 inside Shore 753 chr8 PPM2C NM_018444 inside Shore 43 chr1 KCNJ9 NM_004983 inside Shore 436 chr10 8-Mar NM_001002266 inside Far 69029 chr4 ALPK1 NM_025144 inside Shore 0 chr8 FZD3 NM_017412 downstream Shore 495 chr6 BAT2 NM_004638 inside Shore 52 chr11 HCCA2 NM_053005 inside Far 2351 chr9 MLLT3 NM_004529 inside Shore 509 chr4 C4orf30 NM_017741 downstream Shore 0 chr7 HOXA5 NM_019102 downstream Shore 66 chr13 TMTC4 NM_032813 upstream Far 23274 chr17 SOX9 NM_000346 inside Island 0 chr8 C8ORFK32 NM_015912 inside Shore 258 chr6 KCNK5 NM_003740 inside Shore 640 chr1 SDHC NM_001035512 overlaps 3′ Island 0 chr11 PRDM10 NM_020228 inside Shore 387 chr6 HIST1H3E NM_003532 overlaps 3′ Shore 0 chr10 ZNF438 NM_182755 upstream Shore 35 chr7 KIAA1706 NM_030636 downstream Shore 0 chr12 FBXO21 NM_015002 inside Shore 404 chr12 BCAT1 NM_005504 inside Shore 0 chr19 ZNF599 NM_001007247 inside Shore 126 chr10 HTRA1 NM_002775 inside Shore 437 chr22 C22orf5 NM_012264 promoter Shore 72 chr5 BTF3 NM_001037637 downstream Shore 0 chr2 MTA3 NM_020744 downstream Shore 442 chr5 FER NM_005246 downstream Shore 271 chr2 MYCN NM_005378 upstream cover 0 chr10 INA NM_032727 inside Far 3085 chr19 NOVA2 NM_002516 upstream Far 22266 chr4 SYNPO2 NM_133477 inside Far 52501 chr13 LIG4 NM_002312 overlaps 5′ Shore 0 chr1 LOC148696 NM_001039568 downstream Far 51665 chr8 PPP2R2A NM_002717 inside Shore 1997 chr13 MCF2L NM_024979 inside Shore 1119 chr2 PCGF1 NM_032673 inside Shore 1308 chr12 CLSTN3 NM_014718 inside Far 57129 chr1 PSRC1 NM_001005290 inside Shore 103 chr20 GZF1 NM_022482 downstream Shore 352 chr4 CPE NM_001873 inside Shore 93 chr21 WRB NM_004627 inside Shore 0 chr5 C5orf33 NM_153013 promoter Shore 0 chrX RIBC1 NM_144968 inside Shore 265 chr2 FLJ46838 NM_001007546 inside Shore 243 chrX CDKL5 NM_003159 inside Shore 1550 chr19 IXL NM_017592 overlaps 5′ Shore 718 chr1 C1orf32 NM_199351 inside Shore 159 chr17 SLC39A11 NM_139177 downstream Shore 581 chr17 ENO3 NM_001976 inside Shore 545 chr19 LOC554235 NM_001024656 upstream Shore 875 chr6 WRNIP1 NM_020135 inside Shore 487 chr1 G0S2 NM_015714 downstream Shore 200 chr1 GALNT2 NM_004481 inside Shore 694 chr4 RGS12 NM_198227 overlaps 3′ Far 2387 FDR is false discovery rate. Columns are chromosome, start, end, false discovery rate, tissue-specificity, brain M value, liver M value, spleen M value, gene, annotation, relation to gene, relation to CGI, distance to CGI

TABLE 11 Regions with cancer-specific differential methylation (C-DMRs) at a FDR of 5%. relation Dist to Dist To chr start end deltaM fdr state name annotation region to CGI CGI chr10 5774977 5775480 0.88404 0 Some ASB13 NM_024701 upstream Far 7274 methylation chr11 117907224 117907958 1.00157 0 Some TMEM25 NM_032780 inside Shore 0 methylation chr11 65947037 65948698 −0.88203 0 Less NPAS4 NM_178864 inside Shore 1064 methylation chr13 113615559 113616275 −0.91383 0 Less FAM70B NM_182614 inside cover 0 methylation chr15 58474576 58476390 −0.80517 0 No ANXA2 NM_001002857 inside Shore 651 methylation chr19 5159373 5160095 −1.01444 0 Less PTPRS NM_130854 inside Shore 1469 methylation chr19 5297115 5297780 −0.93292 0 No PTPRS NM_130854 upstream Far 5054 methylation chr19 55631525 55632172 −0.84621 0 Less MYBPC2 NM_004533 inside Far 4036 methylation chr19 60702435 60703541 −0.93061 0 Less NAT14 NM_020378 upstream Far 3082 methylation chr20 33650044 33650445 0.881805 0 More SPAG4 NM_003116 downstream Shore 1688 methylation chr21 37858818 37859555 −1.16368 0 No DYRK1A NM_001396 upstream Shore 402 methylation chr2 147061940 147063253 −0.85834 0 Less ACVR2A NM_001616 downstream Shore 91 methylation chr22 35335058 35336773 −1.03729 0 Less CACNG2 NM_006078 inside Far 44058 methylation chr22 48414225 48414719 −0.96407 0 Less C22orf34 NM_001039473 inside Shore 1806 methylation chr3 193607217 193607552 −0.93573 0 Less FGF12 NM_021032 inside Shore 960 methylation chr3 82629728 82630444 −0.83773 0 Less GBE1 NM_000158 upstream Far 309227 methylation chr4 62618513 62619190 −0.87267 0 Less LPHN3 NM_015236 inside Far 552670 methylation chr5 1718578 1720081 −1.00668 0 Less MRPL36 NM_032479 downstream Shore 106 methylation chr6 52637426 52638797 −0.88756 0 No TMEM14A NM_014051 downstream Shore 0 methylation chr7 153219537 153220325 −1.02674 0 Less DPP6 NM_001039350 inside Far 2938 methylation chr7 27106893 27108170 0.882121 0 More HOXA2 NM_006735 inside Shore 1536 methylation chr8 819298 820365 −0.87447 0 Less ERICH1 NM_207332 upstream Shore 1879 methylation chr9 94989150 94990319 0.845242 0 Some WNK2 NM_006648 inside Shore 1494 methylation chrX 135941512 135943110 0.904877 0 Some GPR101 NM_054021 promoter Island 0 methylation chr13 24843229 24844191 0.835162 2.95E−12 More ATP8A2 NM_016529 downstream Shore 0 methylation chr8 144543158 144544935 −0.79612 3.04E−12 Less RHPN1 NM_052924 upstream Far 9061 methylation chr20 15408602 15408931 −0.87712 5.30E−12 Less C20orf133 NM_001033087 inside Far 1092905 methylation chr19 43436599 43438474 0.828947 6.70E−12 More PPP1R14A NM_033256 inside Shore 4 methylation chr7 27121979 27122917 0.793013 1.34E−11 Some HOXA3 NM_030661 inside Shore 28 methylation chr10 129973472 129974329 −0.79104 1.92E−11 Less MKI67 NM_002417 upstream Far 19204 methylation chr7 129912736 129913242 −0.82465 2.24E−11 Less MEST NM_177524 downstream Shore 11 methylation chr15 90735153 90735977 −0.78099 4.62E−11 Less ST8SIA2 NM_006011 downstream Shore 1704 methylation chr22 36145002 36145832 0.778036 5.64E−11 Some LRRC62 NM_052906 upstream Shore 0 methylation chr5 175019374 175020506 −0.77323 7.72E−11 Less HRH2 NM_022304 downstream Shore 1012 methylation chr8 118032313 118033443 −0.76958 8.22E−11 Less LOC441376 NM_001025357 upstream Far 12173 methylation chr7 27151643 27153628 0.72037 8.57E−11 More HOXA6 NM_024014 inside Shore 0 methylation chr12 108883476 108884015 −0.79921 8.98E−11 Less GIT2 NM_139201 inside Far 34165 methylation chr7 4865828 4866751 −0.75229 1.80E−10 Less PAPOLB NM_020144 inside Shore 1111 methylation chr15 70198422 70199761 −0.75665 2.30E−10 No SENP8 NM_145204 inside Shore 366 methylation chr19 63146186 63149953 −0.69314 2.31E−10 Less ZNF256 NM_005773 inside Shore 545 methylation chr12 128898887 128900474 −0.72032 3.16E−10 No TMEM132D NM_133448 inside Far 5162 methylation chr1 14092469 14093393 0.743518 3.74E−10 More PRDM2 NM_012231 upstream Shore 145 methylation chr20 56858718 56860946 0.682357 3.76E−10 Some GNAS NM_016592 inside cover 0 methylation chr6 50873235 50873811 −0.77277 3.81E−10 Less TFAP2B NM_003221 downstream Far 21434 methylation chr9 101171244 101172098 −0.74221 4.84E−10 Less SEC61B NM_006808 upstream Far 72210 methylation chr7 145026589 145027407 −0.73801 7.53E−10 Less CNTNAP2 NM_014141 downstream Far 416556 methylation chr11 73980533 73981178 −0.78485 8.52E−10 Less POLD3 NM_006591 downstream Shore 0 methylation chr5 16236219 16237106 −0.72798 1.00E−09 No FBXL7 NM_012304 upstream Far 2799 methylation chr9 137437549 137438193 −0.74586 1.27E−09 Less KIAA0649 NM_014811 downstream Far 6096 methylation chr8 17060922 17062917 −0.68442 1.34E−09 Less ZDHHC2 NM_016353 inside Shore 1761 methylation chr9 136758751 136759638 −0.72195 1.46E−09 Less COL5A1 NM_000093 inside Far 40696 methylation chr18 73809395 73809862 −0.76199 1.78E−09 Less GALR1 NM_001480 upstream Far 9620 methylation chr3 129687777 129688211 0.765371 1.99E−09 Some GATA2 NM_032638 inside Shore 0 methylation chr3 191521264 191521946 −0.73631 2.29E−09 No CLDN1 NM_021101 inside Shore 562 methylation chr13 42886712 42887185 −0.75536 2.66E−09 No PIG38 NM_017993 inside Far 370673 methylation chr7 27147025 27148414 0.687423 2.78E−09 More HOXA5 NM_019102 overlaps 3′ Shore 724 methylation chr2 4028054 4028969 −0.71686 2.80E−09 Less ALLC NM_199232 upstream cover 0 methylation chr17 74692697 74693892 −0.70553 2.93E−09 Less LOC146713 NM_001025448 downstream Shore 873 methylation chr12 112557312 112558104 −0.75314 3.06E−09 Less LHX5 NM_022363 upstream Far 43177 methylation chr20 44094989 44097868 −0.67672 3.15E−09 No SLC12A5 NM_020708 inside Shore 634 methylation chr7 92075312 92075873 −0.7558 3.60E−09 Less CDK6 NM_001259 inside Far 17503 methylation chr7 50103520 50103993 −0.74337 5.51E−09 Less ZPBP NM_007009 promoter Shore 53 methylation chr2 172826386 172826961 −0.72901 5.75E−09 No DLX2 NM_004405 upstream Far 18042 methylation chr8 1032896 1034248 −0.68718 5.85E−09 Less ERICH1 NM_207332 upstream Shore 1718 methylation chr20 56841054 56842229 −0.67616 7.76E−09 No GNAS NM_016592 downstream Far 5761 methylation chr7 27129188 27131713 0.62826 7.80E−09 More HOXA3 NM_153631 inside cover 0 methylation chr16 86201868 86205260 −0.67852 9.80E−09 Less JPH3 NM_020655 inside Shore 327 methylation chr18 32022373 32023451 −0.68242 1.01E−08 Less MOCOS NM_017947 inside Shore 230 methylation chr16 31139986 31140921 −0.70124 1.03E−08 Less TRIM72 NM_001008274 inside Far 2105 methylation chrX 23042002 23042577 −0.73787 1.05E−08 Less DDX53 NM_182699 upstream Far 217628 methylation chr12 16648817 16649638 0.693703 1.13E−08 Some LMO3 NM_018640 inside Far 693016 methylation chr11 2190993 2192468 −0.65402 1.36E−08 Less TH NM_000360 upstream Far 46172 methylation chr6 168586102 168588777 0.621865 1.36E−08 More SMOC2 NM_022138 inside Shore 153 methylation chr4 172202321 172202941 −0.71014 1.39E−08 Less GALNT17 NM_001034845 downstream Far 767368 methylation chr20 3603786 3604568 −0.69261 1.43E−08 No ADAM33 NM_025220 inside Shore 1013 methylation chr7 1182527 1183550 −0.70109 1.53E−08 Less ZFAND2A NM_182491 upstream Far 15857 methylation chr2 242632926 242634574 −0.69924 2.62E−08 Less FLJ33590 NM_173821 upstream Shore 1740 methylation chr14 95577239 95578925 −0.64697 3.05E−08 No C14orf132 NM_020215 inside Shore 1084 methylation chr8 966339 968282 −0.62553 3.25E−08 Less ERICH1 NM_207332 upstream Shore 581 methylation chrX 142544143 142544508 −0.72871 3.62E−08 Less SLITRK4 NM_173078 inside Far 4568 methylation chr7 50436454 50438081 −0.63782 3.80E−08 Less IKZF1 NM_006060 overlaps 5′ Shore 560 methylation chr13 87123702 87125149 0.64054 3.98E−08 Some SLITRK5 NM_015567 inside cover 0 methylation chr12 88272781 88274261 −0.65038 4.42E−08 Less DUSP6 NM_001946 upstream Shore 506 methylation chr13 112142200 112143003 −0.6783 4.68E−08 Less C13orf28 NM_145248 upstream Far 11335 methylation chr7 80386676 80389252 −0.60191 4.84E−08 No SEMA3C NM_006379 promoter Shore 14 methylation chr7 90064351 90065062 0.677011 5.06E−08 Some PFTK1 NM_012395 downstream Shore 51 methylation chr8 100031456 100033208 0.618925 5.12E−08 Some OSR2 NM_053001 inside Shore 842 methylation chr13 110126686 110127336 0.682652 5.44E−08 More FLJ12118 NM_024537 inside Shore 1831 methylation chr8 846979 849372 −0.59665 5.47E−08 Less ERICH1 NM_207332 upstream Far 6574 methylation chr11 103540808 103541706 −0.6721 5.56E−08 Less PDGFD NM_025208 upstream Shore 540 methylation chr3 28591265 28591843 0.687621 6.39E−08 Some ZCWPW2 NM_001040432 upstream Shore 0 methylation chr8 98356982 98358151 −0.64161 6.46E−08 Less TSPYL5 NM_033512 inside Shore 629 methylation chr5 3586182 3587073 −0.70608 6.57E−08 Less IRX1 NM_024337 downstream Shore 1557 methylation chr20 4928754 4929383 0.695671 6.64E−08 Some SLC23A2 NM_005116 inside Far 111977 methylation chr12 128900526 128905070 −0.5668 8.89E−08 Less TMEM132D NM_133448 inside Shore 566 methylation chr5 7900127 7901201 −0.65677 9.34E−08 Less FASTKD3 NM_024091 downstream Shore 1744 methylation chr19 35406489 35408138 0.619251 9.87E−08 Some ZNF536 NM_014717 downstream cover 0 methylation chr7 3984090 3985861 −0.6193 9.87E−08 Less SDK1 NM_152744 inside Far 134310 methylation chr18 75366596 75367142 −0.77008 1.07E−07 Less NFATC1 NM_172389 inside Far 4639 methylation chr5 11954677 11955633 −0.64541 1.12E−07 Less CTNND2 NM_001332 inside Shore 917 methylation chr11 2243719 2244678 −0.64164 1.20E−07 Less ASCL2 NM_005170 downstream Far 2002 methylation chr9 137111474 137112189 −0.65985 1.32E−07 Less OLFM1 NM_006334 inside Far 3926 methylation chr7 32076356 32076691 0.709313 1.58E−07 Some PDE1C NM_005020 inside Shore 0 methylation chr11 19323912 19324554 0.68291 1.77E−07 Some E2F8 NM_024680 upstream inside 0 methylation chr20 59905352 59906092 −0.67681 1.89E−07 Less CDH4 NM_001794 inside Shore 1622 methylation chr11 134186796 134187368 −0.6669 2.01E−07 Less B3GAT1 NM_018644 upstream Far 48599 methylation chr7 142264591 142265022 −0.68427 2.20E−07 Less EPHB6 NM_004445 inside Shore 1283 methylation chr6 10501357 10502013 0.657096 2.24E−07 Some TFAP2A NM_001032280 downstream Far 2273 methylation chr7 42233455 42234204 0.645604 2.40E−07 More GLI3 NM_000168 upstream Shore 0 methylation chr12 38783074 38784521 0.600023 2.59E−07 More SLC2A13 NM_052885 inside Shore 708 methylation chr8 117611158 117611838 −0.64748 3.10E−07 Less EIF3S3 NM_003756 downstream Far 225273 methylation chr11 31966319 31967492 −0.63749 3.15E−07 No RCN1 NM_002901 downstream Shore 699 methylation chr17 14831980 14832519 −0.66274 3.17E−07 Less FLJ45831 NM_001001684 upstream Far 272136 methylation chr21 42057731 42059424 −0.58952 3.54E−07 Less RIPK4 NM_020639 inside Shore 0 methylation chr11 133791126 133793856 −0.56461 3.77E−07 No B3GAT1 NM_018644 upstream Far 2997 methylation chr8 22469866 22470840 −0.62573 3.77E−07 Less SORBS3 NM_005775 inside Far 4286 methylation chr3 129686645 129687266 0.658959 3.87E−07 More GATA2 NM_032638 inside Shore 916 methylation chr1 221053963 221054618 −0.65816 4.06E−07 Less FLJ43505 NM_207468 upstream Shore 249 methylation chr10 3499370 3500060 −0.64479 4.34E−07 No PITRM1 NM_014889 upstream Far 8794 methylation chr20 48778557 48780262 −0.59546 4.34E−07 No PARD6B NM_032521 downstream Shore 510 methylation chr7 152249022 152250754 −0.63648 4.67E−07 Less ACTR3B NM_020445 upstream Far 2095 methylation chr12 130718351 130718755 −0.68171 4.82E−07 Less SFRS8 NM_004592 downstream Far 5588 methylation chr14 57669356 57669927 −0.65813 5.16E−07 Less C14orf37 NM_001001872 inside Far 18117 methylation chr10 101273308 101274941 0.602562 5.43E−07 Some NKX2-3 NM_145285 downstream Shore 378 methylation chr18 491107 491721 0.648047 5.52E−07 More COLEC12 NM_130386 upstream Shore 385 methylation chr16 25608094 25609031 −0.61772 5.78E−07 No HS3ST4 NM_006040 downstream Shore 1425 methylation chr19 50669649 50670313 0.642613 5.96E−07 More FOSB NM_006732 overlaps 5′ Shore 1547 methylation chr11 132451977 132454794 −0.55094 6.61E−07 Less OPCML NM_001012393 inside Far 2108 methylation chr13 100427918 100428562 −0.64421 6.75E−07 Less VGCNL1 NM_052867 downstream Far 302194 methylation chr19 4505154 4505870 −0.62946 6.75E−07 Less SEMA6B NM_020241 inside Shore 1066 methylation chr7 139121471 139122220 0.626218 6.75E−07 More TBXAS1 NM_030984 downstream Shore 1655 methylation chr10 1437553 1439093 −0.59022 6.89E−07 Less ADARB2 NM_018702 inside Far 7539 methylation chr13 67580738 67581346 −0.63959 6.96E−07 Less PCDH9 NM_020403 upstream Far 875559 methylation chr3 174341049 174341309 −0.70742 7.33E−07 Less SPATA16 NM_031955 inside Far 254652 methylation chr15 67494729 67495415 −0.63108 7.40E−07 No KIF23 NM_004856 inside Shore 323 methylation chr12 52430235 52431287 0.615375 7.52E−07 Some CALCOCO1 NM_020898 upstream Island 0 methylation chr1 32992444 32992959 0.649479 8.12E−07 Some KIAA1522 NM_020888 inside Shore 0 methylation chr14 52326048 52327349 −0.60366 8.12E−07 No GNPNAT1 NM_198066 inside Shore 65 methylation chr6 80711271 80712497 −0.59439 8.33E−07 Less ELOVL4 NM_022726 inside Shore 966 methylation chr5 132392 134486 −0.57195 8.95E−07 Less KIAA1909 NM_052909 downstream Shore 1942 methylation chr3 148446946 148448043 −0.61085 9.51E−07 Less ZIC4 NM_032153 downstream Far 111759 methylation chr13 113548234 113549273 0.600339 9.66E−07 More GAS6 NM_000820 inside Shore 99 methylation chr11 133445488 133446499 −0.61556 1.00E−06 Less JAM3 NM_032801 inside Shore 597 methylation chr22 47455137 47456387 −0.58401 1.05E−06 Less FAM19A5 NM_015381 inside Far 4801 methylation chr8 132984463 132985363 −0.6234 1.10E−06 Less KIAA0143 NM_015137 downstream Shore 141 methylation chr2 100303400 100304122 0.61944 1.14E−06 More LONRF2 NM_198461 upstream Shore 89 methylation chr17 22902924 22903499 −0.63407 1.14E−06 Less KSR1 NM_014238 inside Far 53125 methylation chr1 206062462 206063842 0.573914 1.22E−06 Some LOC148696 NM_001039568 overlaps 5′ Far 44643 methylation chr5 134553843 134554653 0.610755 1.28E−06 More H2AFY NM_138609 downstream Shore 173 methylation chr22 46984389 46985561 −0.58562 1.31E−06 Less LOC388915 NM_001010902 upstream Shore 218 methylation chr19 40896785 40897432 0.623253 1.34E−06 More ZBTB32 NM_014383 inside Shore 1492 methylation chr7 149667145 149668311 0.582745 1.37E−06 More RARRES2 NM_002889 inside Shore 81 methylation chr4 81341228 81342411 0.593058 1.40E−06 More PRDM8 NM_020226 inside Shore 121 methylation chr12 4889976 4890592 0.629828 1.42E−06 Some KCNA1 NM_000217 downstream inside 0 methylation chr19 36535109 36535822 0.615047 1.43E−06 More TSHZ3 NM_020856 upstream Shore 0 methylation chr10 24025406 24026498 −0.60568 1.43E−06 No C10orf67 NM_153714 upstream Shore 422 methylation chr19 38866174 38866577 −0.67497 1.47E−06 Less CHST8 NM_022467 downstream Shore 607 methylation chr19 16876414 16876750 −0.6835 1.49E−06 Less CPAMD8 NM_015692 inside Far 6557 methylation chr11 31780765 31782078 0.584509 1.55E−06 Some PAX6 NM_000280 inside Shore 241 methylation chr11 45645190 45645996 −0.6053 1.70E−06 Less CHST1 NM_003654 upstream Shore 1119 methylation chr13 21140646 21141497 0.598118 1.82E−06 Some FGF9 NM_002010 downstream Shore 0 methylation chr8 53636369 53637193 −0.60042 1.87E−06 Less UNQ9433 NM_207413 inside Far 2904 methylation chr10 102097628 102098179 −0.64186 2.02E−06 No SCD NM_005063 inside Shore 0 methylation chr13 109229161 109231936 −0.52866 2.19E−06 Less IRS2 NM_003749 inside Shore 531 methylation chr15 53707187 53707654 −0.63505 2.19E−06 Less PRTG NM_173814 inside Far 38667 methylation chr11 1861758 1862726 −0.58891 2.22E−06 Less LSP1 NM_001013255 inside Shore 1848 methylation chr20 24399098 24400197 0.576246 2.35E−06 Some C20orf39 NM_024893 inside Island 0 methylation chr13 113850874 113851696 0.595891 2.36E−06 More RASA3 NM_007368 inside Far 2472 methylation chr6 7413675 7414250 −0.61922 2.43E−06 Less RIOK1 NM_153005 upstream Far 72267 methylation chr10 124898365 124898865 0.631343 2.63E−06 Some HMX2 NM_005519 inside inside 0 methylation chr7 5360462 5361424 0.593453 2.65E−06 More SLC29A4 NM_153247 upstream Shore 974 methylation chr14 100996473 100997806 −0.5653 2.71E−06 Less DIO3 NM_001362 downstream Shore 725 methylation chr19 13473726 13475470 −0.56537 2.71E−06 Less CACNA1A NM_023035 inside Far 2282 methylation chr8 3257254 3257862 −0.61199 2.86E−06 Less CSMD1 NM_033225 inside Far 389899 methylation chr16 49141667 49142498 −0.59792 2.87E−06 Less NKD1 NM_033119 inside Shore 701 methylation chr5 158465126 158466712 0.548688 2.93E−06 Some EBF1 NM_024007 upstream cover 0 methylation chr19 3240846 3241491 −0.60751 2.96E−06 Less BRUNOL5 NM_021938 inside Far 2776 methylation chr6 5946646 5947326 0.603987 2.96E−06 Some NRN1 NM_016588 inside Shore 144 methylation chr7 154079253 154079678 −0.65898 3.29E−06 Less DPP6 NM_001936 inside Far 93314 methylation chr8 144372893 144373912 −0.59142 3.41E−06 No LOC338328 NM_178172 upstream Far 9171 methylation chr16 4101996 4102769 0.594061 3.47E−06 Some ADCY9 NM_001116 inside Far 2040 methylation chr20 61186086 61186916 −0.58801 3.49E−06 Less BHLHB4 NM_080606 upstream Shore 1296 methylation chr1 203580235 203580810 0.611495 3.58E−06 More KLHDC8A NM_018203 inside Shore 36 methylation chr2 128944074 128944714 −0.61563 3.59E−06 Less HS6ST1 NM_004807 upstream Shore 357 methylation chr20 48848947 48849522 0.611074 3.65E−06 More BCAS4 NM_017843 inside Far 3398 methylation chr18 75183050 75183445 0.635066 3.79E−06 More ATP9B NM_198531 inside Far 5006 methylation chr6 133602937 133603652 0.618352 3.94E−06 More EYA4 NM_172103 downstream Shore 127 methylation chr9 97304533 97306736 0.542219 4.02E−06 More PTCH1 NM_000264 inside Shore 1560 methylation DeltaM is cancer minus normal. FDR is false discovery rate. State: “Some methylation” means some methylation in tumor, none in normal; “Less methylation” means less in tumor than normal. “More methylation” means more in tumor than normal; “No methylation” means none in tumor, some in normal. Columns are chromosome, start, end, delta M, fdr, overall methylation state, gene, annotation, relation to gene, relation to CGI, distance to CGI

TABLE 12 Relationship between tissue methylation-specific gene expression and experimental demethylation TISSUES Gene Mean expression AZA/HCT116 AZA P- TISSUES_Diff_expression expression Description (AZA) meanHCT116 (mean) AZA_expression_direction value (L − B) P-value MEF2C 1.50 0.94 1.60 increase 0.0167 −5.4 1.10E−05 CCK 1.68 0.96 1.74 increase 0.0256 −5.2 4.50E−05 NTRK2 2.11 0.95 2.23 increase 0.0167 −4.7 4.40E−05 SNCA 0.86 0.52 1.67 increase 0.0256 −4.3 6.30E−05 HSPA8 5.33 2.89 1.85 increase 0.0167 −3.3 0.00053 WRB 0.88 0.56 1.57 increase 0.0256 −2.2 0.00037 PIN 1.17 0.67 1.75 increase 0.0167 −1.8 6.90E−05 SOX9 2.78 1.81 1.53 increase 0.0167 −1.6 0.0042 KAL1 1.79 1.04 1.73 increase 0.0256 −1.5 0.00058 FYN 0.91 0.58 1.57 increase 0.0167 −1.5 0.0071 ABCC5 1.49 0.90 1.65 increase 0.0256 −1.5 4.20E−05 MTMR6 1.72 1.11 1.55 increase 0.0476 −1 0.0039 SHC1 1.99 1.17 1.70 increase 0.0167 1 8.00E−04 S100A10 1.27 0.84 1.51 increase 0.0167 1.1 0.0014 IFITM3 2.65 1.26 2.11 increase 0.0256 1.2 0.056 IFITM3 3.12 1.05 2.97 increase 0.0167 1.2 0.056 DUSP6 6.10 3.29 1.85 increase 0.0167 1.3 0.004 ACADVL 1.71 1.12 1.53 increase 0.0167 1.3 0.012 MT2A 3.31 2.20 1.50 increase 0.0256 1.6 0.044 KCNJ8 2.57 1.23 2.08 increase 0.0455 1.7 0.00052 MT1X 3.15 2.08 1.52 increase 0.0167 1.9 0.052 NFKBIA 2.04 1.07 1.91 increase 0.0167 2.2 0.00014 SDC4 2.88 1.48 1.95 increase 0.0167 2.2 0.004 CD14 1.75 0.82 2.14 increase 0.0455 2.4 0.0081 EGFR 2.02 1.12 1.81 increase 0.0256 2.5 0.00029 GLDC 1.32 0.70 1.88 increase 0.0256 2.7 0.00087 IGFBP3 0.44 0.04 11.24 increase 0.0167 2.8 2.70E−05 GCH1 1.14 0.73 1.55 increase 0.0167 3.3 0.0069 Gene TISSUES_deltaM TISSUES_Methylation T-DMR Description TISSUES_expression_direction_in_hypomethylated_tissue (L − B) FDR Location MEF2C increase 0.74 3.60E−05 Far CCK increase 0.84 4.90E−05 Shore NTRK2 increase 0.48 0.0089 Far SNCA increase 0.36 0.0089 Shore HSPA8 increase 0.42 0.0089 Shore WRB decrease −0.52 0.0089 Shore PIN increase 1.1 1.00E−07 Shore SOX9 increase 0.77 0.00035 Shore KAL1 increase 0.55 0.0017 Shore FYN increase 0.6 0.0012 Shore ABCC5 increase 0.93 1.40E−08 Shore MTMR6 increase 0.79 0.00025 Far SHC1 decrease 1 5.50E−07 Far S100A10 increase −0.49 0.0089 Shore IFITM3 increase −0.56 0.0045 Shore IFITM3 increase −0.56 0.0045 Shore DUSP6 decrease −1.4 9.90E−14 Far ACADVL decrease 0.62 0.0012 Far MT2A increase −0.91 8.40E−08 Shore KCNJ8 increase −0.58 0.0077 Shore MT1X increase −0.46 0.0089 Shore NFKBIA increase −0.69 0.00097 Far SDC4 increase −0.44 0.0089 Shore CD14 increase −0.46 0.0056 Shore EGFR increase −0.67 3.00E−04 Shore GLDC increase −0.81 0.00058 Shore IGFBP3 increase 0.48 0.0058 Shore GCH1 increase −0.54 0.00053 Shore Data for AZA and HCT116 gene expression data was obtained from Gius et al. (Gius et al., Cancer Cell, 6: 361-71, 2004). AZA represents expression data from HCT116 cells treated with 5-aza-2′-deoxycytidine TISSUES represents data (methylation and expression) from liver and brain samples examined in the current study. L = liver, B = brain, deltaM is differential methylation

TABLE 13 Relationship between tissue methylation-specific gene expression and experimental demethylation TISSUES_Diff_expression TISSUES Gene Mean DKO/HCT116 DKO p- (L − expression Description expression (DKO) meanHCT116 (mean) DKO_expression_direction value B) P-value BASP1 1.2362 0.5882 2.1017 increased 0.0014 −5.3 6.10E−07 TUBA1A 0.9824 0.4569 2.1499 increased 0.0001 −4.1 3.70E−06 TUBB2B 1.6697 0.5768 2.8949 increased 0 −3.9 2.30E−05 PRKAR2B 1.4493 0.896 1.6175 increased 0.0064 −3.5 2.00E−05 TUBB4 2.0428 0.8001 2.5532 increased 0.0057 −3.4 2.90E−05 YWHAE 1.5439 1.0142 1.5222 increased 0.0276 −2.9 7.60E−05 YWHAQ 1.6098 0.7673 2.098 increased 0.0191 −2.7 0.00011 NDUFA5 1.0375 0.6292 1.6489 increased 0.0198 −2.5 0.006 MAPRE2 1.9434 1.2365 1.5717 increased 0.0264 −2.4 8.40E−06 HSPA8 1.3072 0.7291 1.7929 increased 0.009 −2.2 0.0015 NTRK2 1.4017 0.8025 1.7468 increased 0.008 −2.2 0.0041 STMN1 1.9323 0.9595 2.0139 increased 0.0002 −2.2 0.00012 PPP2CA 1.905 1.1691 1.6295 increased 0.0476 −2.1 0.0024 APPBP2 1.8646 1.2318 1.5138 increased 0.0028 −1.9 4.60E−05 DCK 1.4481 0.85 1.7038 increased 0.0003 −1.9 0.00097 PIN1 1.5581 0.6673 2.3351 increased 0 −1.8 6.90E−05 PPP1CB 2.218 1.3206 1.6796 increased 0.0384 −1.5 0.0094 AP1S2 1.8289 1.0252 1.7839 increased 0.0331 −1.5 0.00045 TPI1 1.1161 0.6111 1.8266 increased 0.0001 −1.4 0.00055 AP2B1 1.8708 1.1524 1.6234 increased 0.0106 −1.4 4.50E−05 FGF12 1.0432 0.5635 1.8512 increased 0.0021 −1.3 0.0048 SGCE 1.6516 0.7029 2.3498 increased 0.0001 −1.2 0.00054 CAP1 1.6185 1.021 1.5852 increased 0.0163 −1.2 0.0068 H2AFX 1.5148 0.954 1.5878 increased 0.0017 −1.1 0.00058 RAD51C 2.0561 1.1202 1.8354 increased 0.0002 −1 0.0017 Gene TISSUES_deltaM TISSUES_Methylation T-DMR Description TISSUES_expression_direction_in_hypomethylated_tissue (L − B) FDR Location BASP1 increased 0.5 0.0089 Shore TUBA1A increased 0.69 0.00047 Shore TUBB2B increased 0.53 0.0089 Shore PRKAR2B increased 0.66 0.0089 Far TUBB4 increased 0.61 0.00067 Shore YWHAE increased 0.63 0.003 Shore YWHAQ increased 0.52 0.0048 Shore NDUFA5 increased 0.66 0.0037 Shore MAPRE2 increased 0.53 0.0089 Shore HSPA8 increased 0.42 0.0089 Shore NTRK2 increased 0.48 0.0089 Far STMN1 increased 0.93 3.50E−07 Shore PPP2CA increased 0.66 8.70E−05 Shore APPBP2 increased 0.4 0.0089 Shore DCK increased 0.54 0.0089 Far PIN1 increased 1.1 1.00E−07 Shore PPP1CB increased 0.54 0.0089 Shore AP1S2 increased 0.63 0.0089 Shore TPI1 increased 0.45 0.0089 Shore AP2B1 increased 0.5 0.0089 Shore FGF12 increased 0.67 0.00031 Shore SGCE increased 0.35 0.0089 Shore CAP1 increased 0.51 0.0089 Shore H2AFX increased 0.62 0.0013 Shore RAD51C increased 0.59 0.0069 Shore Data for DKO and HCT116 gene expression data was obtained from Gius et al. (Gius et al., Cancer Cell, 6: 361-71, 2004). DKO represents expression data from HCT116 cells with a genetic knockout of DNA methylatransferases 1 and 3b TISSUES represents data (methylation and expression) from liver and brain samples examined in the current study. L = liver, B = brain, deltaM is differential methylation

TABLE 14 Regions with tissue-specific differential methylation (T-DMRs) and differential methylation in colon cancer (C- DMRs) at a FDR of 5%. Dist chr start end tumorstate similartissue name annotation region island To CGI chr10 5774977 5775480 Some brain ASB13 NM_024701 upstream Far 7274 methylation chr21 37858818 37859555 No brain DYRK1A NM_001396 upstream Shore 402 methylation chr11 73980533 73981178 Less brain POLD3 NM_006591 downstream Shore 0 methylation chr7 92075312 92075873 Less brain CDK6 NM_001259 inside Far 17503 methylation chr6 168586102 168588777 More brain SMOC2 NM_022138 inside Shore 153 methylation chr9 137111474 137112189 Less brain OLFM1 NM_006334 inside Far 3926 methylation chr7 42233455 42234204 More brain GLI3 NM_000168 upstream Shore 0 methylation chr1 221053963 221054618 Less brain FLJ43505 NM_207468 upstream Shore 249 methylation chr10 3499370 3500060 No brain PITRM1 NM_014889 upstream Far 8794 methylation chr19 4505154 4505870 Less brain SEMA6B NM_020241 inside Shore 1066 methylation chr12 52430235 52431287 Some brain CALCOCO1 NM_020898 upstream Island 0 methylation chr6 80711271 80712497 Less brain ELOVL4 NM_022726 inside Shore 966 methylation chr8 132984463 132985363 Less brain KIAA0143 NM_015137 downstream Shore 141 methylation chr7 149667145 149668311 More brain RARRES2 NM_002889 inside Shore 81 methylation chr19 13473726 13475470 Less brain CACNA1A NM_023035 inside Far 2282 methylation chr8 3257254 3257862 Less brain CSMD1 NM_033225 inside Far 389899 methylation chr20 48848947 48849522 More brain BCAS4 NM_017843 inside Far 3398 methylation chr6 133602937 133603652 More brain EYA4 NM_172103 downstream Shore 127 methylation chr17 41333049 41334735 Less brain MAPT NM_005910 inside Far 2213 methylation chr9 73954546 73955019 No brain GDA NM_004293 inside Shore 0 methylation chr20 33336261 33337040 No brain FAM83C NM_178468 overlaps 3′ Shore 108 methylation chr1 20383082 20384429 Less brain UBXD3 NM_152376 downstream Shore 518 methylation chr11 64244495 64245421 Less brain NRXN2 NM_015080 inside Shore 1648 methylation chr7 121736808 121737526 Some brain FEZF1 NM_001024613 upstream Shore 0 methylation chr13 20184958 20186542 No brain IL17D NM_138284 inside Far 7069 methylation chr10 112248574 112249464 Some brain DUSP5 NM_004419 inside Shore 0 methylation chrX 120009921 120011075 Less brain GLUD2 NM_012084 inside Shore 270 methylation chr17 8846444 8846986 Some brain NTN1 NM_004822 downstream Shore 0 methylation chrX 74062751 74063503 Less brain KIAA2022 NM_001008537 upstream Shore 850 methylation chr7 142205460 142206135 Some brain PRSS2 NM_002770 upstream Shore 84 methylation chr15 76344533 76345543 Less brain DNAJA4 NM_018602 inside Shore 0 methylation chr7 101853738 101854712 More brain PRKRIP1 NM_024653 overlaps 5′ Far 5898 methylation chr14 102437341 102437847 More brain TRAF3 NM_003300 inside Far 5743 methylation chr9 37022893 37023922 More brain PAX5 NM_016734 inside Shore 213 methylation chr10 3818645 3819574 Less brain KLF6 NM_001300 upstream Shore 519 methylation chr7 12694278 12696066 No brain ARL4A NM_005738 inside Shore 505 methylation chr20 47529867 47531270 No brain KCNB1 NM_004975 inside Shore 649 methylation chr20 49590611 49591553 Some brain NFATC2 NM_173091 inside Shore 758 methylation chr9 122729717 122730552 More brain TRAF1 NM_005658 upstream Shore 40 methylation chr10 22807699 22808944 Less brain PIP5K2A NM_005028 downstream Shore 643 methylation chr7 38634847 38635894 Less brain AMPH NM_001635 inside Shore 1070 methylation chr12 55902867 55903735 More brain NXPH4 NM_007224 inside Shore 1301 methylation chr19 47263128 47264262 No brain GRIK5 NM_002088 upstream Far 7513 methylation chr18 68357323 68359390 No brain CBLN2 NM_182511 inside Shore 564 methylation chr7 92076105 92076398 No brain CDK6 NM_001259 inside Far 18296 methylation chr15 88348758 88349331 Less brain ZNF710 NM_198526 inside Shore 1843 methylation chr19 46525737 46526942 Some brain TGFB1 NM_000660 downstream Shore 1912 methylation chr8 10955476 10957641 Less brain XKR6 NM_173683 inside Shore 969 methylation chr7 150958618 150959472 Less brain PRKAG2 NM_024429 inside Far 26959 methylation chr10 49550539 49551086 More brain ARHGAP22 NM_021226 upstream Far 15932 methylation chr11 63821175 63822065 Some brain KCNK4 NM_033310 inside Shore 1268 methylation chr16 34067297 34067906 Less brain LOC649159 NM_001040069 upstream Shore 548 methylation chr7 5224659 5225234 More brain WIPI2 NM_001033520 inside Far 8213 methylation chr19 14487242 14488698 Less brain DNAJB1 NM_006145 inside Shore 708 methylation chr6 160690246 160691751 Less brain SLC22A3 NM_021977 inside Shore 0 methylation chr19 2239906 2240688 More brain C19orf35 NM_198532 upstream Shore 207 methylation chr10 103579583 103580518 Some brain KCNIP2 NM_173194 inside Island 0 methylation chr7 121738145 121739034 Some brain FEZF1 NM_001024613 upstream Shore 0 methylation chr13 101842566 101843905 No brain FGF14 NM_175929 inside Shore 776 methylation chr2 935104 935722 Some brain SNTG2 NM_018968 downstream Shore 0 methylation chr2 88531875 88532417 More brain FLJ25369 NM_152670 downstream Shore 33 methylation chr5 1555785 1556980 More brain AYTL2 NM_024830 inside Far 7498 methylation chr11 66892179 66893553 More brain CLCF1 NM_013246 inside Far 2910 methylation chr10 23425480 23426204 No brain MSRB2 NM_012228 inside Shore 396 methylation chr3 5002243 5003439 Less brain BHLHB2 NM_003670 upstream Shore 0 methylation chr2 174898625 174899336 More brain FLJ46347 NM_001005303 downstream Shore 0 methylation chr19 37859830 37860375 More brain RGS9BP NM_207391 inside Shore 0 methylation chr15 91430517 91431653 No brain RGMA NM_020211 inside Shore 779 methylation chr10 101283725 101284669 Some brain NKX2-3 NM_145285 inside Shore 0 methylation chr20 9436915 9438354 More brain C20orf103 NM_012261 downstream Shore 1026 methylation chr15 28985194 28985874 More brain KIAA1018 NM_014967 inside Shore 1510 methylation chr1 57861340 57861987 Less brain DAB1 NM_021080 inside Far 198115 methylation chr1 55123414 55124391 Less brain DHCR24 NM_014762 inside Shore 660 methylation chr18 22385126 22385774 Some brain KCTD1 NM_198991 inside Shore 0 methylation chr13 111762553 111763273 Some brain SOX1 NM_005986 downstream Shore 87 methylation chr10 124218023 124218949 Less brain HTRA1 NM_002775 inside Far 5793 methylation chr13 99424284 99425382 Some brain ZIC5 NM_033132 upstream Shore 1935 methylation chr13 36147816 36148563 Less brain LOC400120 NM_203451 inside Shore 1353 methylation chr8 120755795 120756355 Less brain ENPP2 NM_001040092 upstream Far 157624 methylation chr8 145074517 145075008 More brain PLEC1 NM_201384 inside Shore 394 methylation chr1 35164297 35165947 Less brain ZMYM6 NM_007167 downstream Shore 1387 methylation chr5 72958425 72958969 Less brain UTP15 NM_032175 upstream Shore 142 methylation chr18 42166935 42167750 Some brain RNF165 NM_152470 downstream Shore 0 methylation chr15 90198817 90201147 Some brain SLCO3A1 NM_013272 inside Shore 131 methylation chr13 25694712 25696110 No brain RNF6 NM_005977 promoter Shore 0 methylation chr10 121290732 121291137 More brain RGS10 NM_001005339 inside Shore 331 methylation chr10 119281507 119283969 Some brain EMX2 NM_004098 downstream Shore 0 methylation chr2 45010614 45011650 Some brain SIX3 NM_005413 downstream Shore 61 methylation chr10 101831054 101831872 Less brain CPN1 NM_001308 overlaps 5′ Far 15878 methylation chr7 2305691 2306476 More brain SNX8 NM_013321 inside Far 13679 methylation chr20 54635841 54636933 Some brain TFAP2C NM_003222 downstream inside 0 methylation chr7 102577345 102577848 No brain NAPE-PLD NM_198990 upstream Shore 0 methylation chr20 54632900 54633442 Some brain TFAP2C NM_003222 downstream Shore 244 methylation chr6 106657399 106658118 Some brain PRDM1 NM_182907 inside Far 16321 methylation chr10 89410644 89411916 Less brain PAPSS2 NM_001015880 inside Shore 637 methylation chr5 139705099 139705722 No brain HBEGF NM_001945 inside Shore 0 methylation chr7 55344238 55344779 More brain LANCL2 NM_018697 downstream Far 34680 methylation chr19 45414041 45414847 Less brain TTC9B NM_152479 inside Shore 97 methylation chr16 71648030 71648287 Some brain ATBF1 NM_006885 upstream Shore 728 methylation chr3 184362174 184362794 More brain LAMP3 NM_014398 inside Shore 174 methylation chr9 2232790 2233418 No brain SMARCA2 NM_003070 upstream Shore 688 methylation chr17 35277006 35277374 More brain ZPBP2 NM_198844 downstream Shore 297 methylation chr2 27336168 27336881 Less brain SLC30A3 NM_003459 inside Shore 1496 methylation chr6 170702309 170703586 Less brain PSMB1 NM_002793 inside Shore 463 methylation chr2 100089371 100089913 Less brain AFF3 NM_002285 inside Far 2198 methylation chr13 32487129 32487948 More brain KL NM_153683 downstream Shore 0 methylation chr16 88601552 88602716 More brain DBNDD1 NM_024043 inside Far 3168 methylation chr17 40746938 40747474 Some brain MAP3K14 NM_003954 inside Far 2200 methylation chr7 76881860 76882613 More brain LOC54103 NM_017439 upstream Shore 163 methylation chr9 109287301 109288864 Some brain KLF4 NM_004235 inside Shore 705 methylation chr19 36536018 36538335 Some brain TSHZ3 NM_020856 upstream Shore 0 methylation chr19 1811760 1812617 No brain KLF16 NM_031918 inside Shore 94 methylation chr7 44051835 44052197 Some brain DBNL NM_001014436 inside Shore 563 methylation chr13 111760735 111762537 Some brain SOX1 NM_005986 downstream Shore 69 methylation chr1 243387598 243388628 More brain EFCAB2 NM_032328 upstream Shore 728 methylation chr11 67008185 67008540 More brain AIP NM_003977 inside Shore 745 methylation chr1 3816414 3816740 More brain C1orf174 NM_207356 upstream Far 2098 methylation chr20 41978520 41978954 Some brain C20orf100 NM_032883 inside Shore 0 methylation chr4 54663443 54664534 Some brain GSH2 NM_133267 upstream Shore 623 methylation chr8 101641754 101641969 Less brain ANKRD46 NM_198401 upstream Shore 474 methylation chr6 10506685 10507236 More brain TFAP2A NM_001032280 inside Shore 0 methylation chr9 125811111 125813109 Some brain LHX2 NM_004789 downstream Island 0 methylation chr2 71358949 71359598 Less brain ZNF638 NM_001014972 downstream Shore 1208 methylation chr2 50910673 50911104 Less brain NRXN1 NM_004801 inside Far 196922 methylation chr11 75599856 75600608 More brain WNT11 NM_004626 upstream Island 0 methylation chr13 111776858 111777751 Some brain SOX1 NM_005986 upstream Shore 438 methylation chr8 133756854 133758207 No brain LRRC6 NM_012472 overlaps 5′ Island 0 methylation chr21 43964727 43965443 Less brain PDXK NM_003681 inside Shore 468 methylation chr2 65072672 65073755 Less brain SLC1A4 NM_003038 inside Shore 1956 methylation chr12 112388093 112389548 Some brain LHX5 NM_022363 inside inside 0 methylation chr11 128070356 128071042 Some brain FLI1 NM_002017 inside Shore 135 methylation chr7 29814368 29815975 Less brain SCRN1 NM_014766 downstream Shore 1092 methylation chr13 111767784 111768464 Some brain SOX1 NM_005986 downstream Shore 101 methylation chr1 110412699 110412994 More brain ALX3 NM_006492 inside inside 0 methylation chr15 87749926 87751409 Some brain RHCG NM_016321 downstream Island 0 methylation chr21 45692055 45692558 Less brain COL18A1 NM_130445 inside Far 6999 methylation chr4 42096924 42097709 More brain ATP8A1 NM_006095 downstream Shore 1365 methylation chr10 119295529 119296699 Some brain EMX2 NM_004098 inside Shore 171 methylation chr18 9902703 9903140 Less brain VAPA NM_194434 downstream Shore 324 methylation chr11 119937785 119939272 Less brain ARHGEF12 NM_015313 upstream Shore 760 methylation chr13 66699432 66700406 Less brain PCDH9 NM_020403 inside Far 2188 methylation chr21 45319964 45320506 Some brain ADARB1 NM_001033049 inside Shore 64 methylation chr8 11586977 11587378 Some brain GATA4 NM_002052 downstream Shore 0 methylation chr14 34941365 34942010 Some brain NFKBIA NM_020529 inside Shore 788 methylation chr9 963736 964822 Some brain DMRT3 NM_021240 downstream Shore 460 methylation chr11 7489950 7490879 Some brain PPFIBP2 NM_003621 downstream Shore 251 methylation chr9 135226940 135227305 More brain SURF4 NM_033161 inside Far 4822 methylation chr3 195887111 195887789 More brain FAM43A NM_153690 downstream Shore 0 methylation chr11 62449546 62449908 More brain CHRM1 NM_000738 upstream Shore 41 methylation chr7 5237174 5238382 More brain WIPI2 NM_001033520 inside Far 2538 methylation chr1 119335409 119336717 Some brain TBX15 NM_152380 upstream Shore 472 methylation chr7 157176767 157177234 Some brain PTPRN2 NM_002847 inside inside 0 methylation chr7 99059893 99060630 More brain ZNF498 NM_145115 inside Far 7072 methylation chr4 99799616 99800611 No brain TSPAN5 NM_005723 upstream Shore 433 methylation chr1 163470852 163471256 Some brain LMX1A NM_177398 inside Shore 0 methylation chr14 72673969 72674259 Less brain PSEN1 NM_000021 inside Shore 469 methylation chr15 50862519 50863517 More brain ONECUT1 NM_004498 inside Shore 0 methylation chr7 157748940 157749938 More brain PTPRN2 NM_002847 inside Shore 1574 methylation chr5 125957145 125958451 No brain ALDH7A1 NM_001182 inside Shore 49 methylation chr9 137135684 137136292 Less brain OLFM1 NM_014279 inside Shore 899 methylation chr3 193611789 193611977 Less brain FGF12 NM_004113 inside Shore 1104 methylation chr3 10834684 10835283 Less brain SLC6A11 NM_014229 inside Shore 1237 methylation chr3 35654544 35655159 Less brain ARPP-21 NM_016300 downstream Shore 354 methylation chr4 54669495 54670070 More brain GSH2 NM_133267 upstream Shore 74 methylation chr11 71467385 71467930 No brain NUMA1 NM_006185 inside Shore 1103 methylation chr20 61838213 61838785 Some brain LIME1 NM_017806 overlaps 3′ Shore 614 methylation Columns are chromosome, start, end, methylation level of tumor, tissue-specificity, gene, annotation, relation to gene, relation to CGI, distance to CGI

TABLE 15 Gene ontology functional categories enriched in hypermethylated C-DMRs (P < 0.01) GOBPID Pvalue OddsRatio ExpCount Count Size Term Region GO: 0065007 1.75E−15 1.434797 1049.09 1221 3840 biological inside regulation GO: 0006355 2.40E−13 1.500956 504.3282 632 1846 regulation of inside/promoter transcription, DNA-dependent GO: 0022008 1.49E−12 2.556799 66.11453 117 242 neurogenesis inside/promoter GO: 0032774 2.03E−12 1.47175 517.4418 641 1894 RNA biosynthetic inside/promoter process GO: 0019219 1.13E−11 1.442249 545.5814 667 1997 regulation of inside/promoter nucleobase, nucleoside, nucleotide and nucleic acid metabolic process GO: 0006350 2.42E−11 1.431476 552.1383 672 2021 transcription inside/promoter GO: 0010468 9.44E−11 1.410716 569.6231 687 2085 regulation of inside gene expression GO: 0007399 3.96E−10 2.235828 69.90378 116 263 nervous system inside development GO: 0019222 7.12E−10 1.374211 622.0776 737 2277 regulation of inside metabolic process GO: 0007275 2.63E−09 1.905658 100.0965 152 395 multicellular inside/promoter organismal development GO: 0050794 2.78E−09 1.36836 612.7722 721 2297 regulation of inside cellular process GO: 0007155 3.54E−09 1.735154 143.9767 204 527 cell adhesion inside GO: 0048667 1.03E−08 3.004827 30.59846 59 112 neuron inside/promoter morphogenesis during differentiation GO: 0031175 2.06E−08 2.788835 33.87686 63 124 neurite inside/promoter development GO: 0006366 5.56E−07 1.574135 154.0851 206 564 transcription inside from RNA polymerase II promoter GO: 0048598 6.02E−07 3.034178 22.67564 44 83 embryonic inside/promoter morphogenesis GO: 0006813 7.63E−07 2.392713 36.60887 63 134 potassium ion inside transport GO: 0048731 2.51E−06 1.509823 163.7593 214 629 system inside development GO: 0007268 4.80E−06 2.061921 46.00111 73 169 synaptic inside transmission GO: 0001501 6.53E−06 2.073943 43.98528 70 161 skeletal inside development GO: 0007166 7.82E−06 1.380202 258.195 316 947 cell surface inside/promoter receptor linked signal transduction GO: 0048663 8.67E−06 16.02797 3.824807 12 14 neuron fate inside/promoter commitment GO: 0009887 1.12E−05 1.725707 77.15602 110 283 organ inside morphogenesis GO: 0048858 1.20E−05 1.975596 47.81009 74 175 cell projection inside/promoter morphogenesis GO: 0007411 1.37E−05 3.379597 14.20643 29 52 axon guidance inside GO: 0032989 1.71E−05 1.806973 62.01652 91 227 cellular structure inside morphogenesis GO: 0007417 1.77E−05 4.033628 4.162579 15 210 central nervous promoter/inside system development GO: 0045165 1.78E−05 5.117343 7.884457 19 29 cell fate inside commitment GO: 0050877 3.16E−05 1.457508 153.5387 196 562 neurological inside system process GO: 0030182 4.77E−05 4.13478 3.500752 13 180 neuron promoter/inside differentiation GO: 0003002 6.81E−05 3.011824 14.43907 28 53 regionalization inside GO: 0030198 7.50E−05 4.234686 8.469216 19 31 extracellular inside matrix organization and biogenesis GO: 0007165 0.000112 1.27058 370.8697 428 1393 signal inside transduction GO: 0007498 0.000119 3.462613 10.65482 22 39 mesoderm inside development GO: 0006811 0.000179 1.761983 50.34717 73 186 ion transport inside/promoter GO: 0016481 0.000212 1.621181 69.11973 95 253 negative inside regulation of transcription GO: 0008286 0.000212 4.1309 7.649615 17 28 insulin receptor inside signaling pathway GO: 0007223 0.00022 5.786819 5.19081 13 19 Wnt receptor inside signaling pathway, calcium modulating pathway GO: 0048518 0.000234 1.314643 236.0452 281 864 positive inside regulation of biological process GO: 0007156 0.00028 1.973959 32.23766 50 118 homophilic cell inside adhesion GO: 0007409 0.000289 2.697344 14.67244 27 54 axonogenesis inside/promoter GO: 0006928 0.000332 1.539749 81.96016 109 300 cell motility inside GO: 0000122 0.000357 1.94508 32.51086 50 119 negative inside regulation of transcription from RNA polymerase II promoter GO: 0051253 0.000364 1.720385 49.72249 71 182 negative inside regulation of RNA metabolic process GO: 0048869 0.000389 1.879478 26.48193 44 1336 cellular promoter developmental process GO: 0030901 0.000399 30.11077 0.158574 3 8 midbrain promoter/inside development GO: 0019933 0.000447 2.434235 17.21163 30 63 cAMP-mediated inside signaling GO: 0048523 0.000503 1.381743 142.3193 176 525 negative inside regulation of cellular process GO: 0009790 0.000527 3.008165 10.78958 21 40 embryonic inside development GO: 0007169 0.000542 3.862582 2.834518 10 143 transmembrane promoter/inside receptor protein tyrosine kinase signaling pathway GO: 0045941 0.000581 1.568199 68.30013 92 250 positive inside regulation of transcription GO: 0035107 0.000665 3.174262 9.562018 19 35 appendage inside/promoter morphogenesis GO: 0060173 0.000665 3.174262 9.562018 19 35 limb inside/promoter development GO: 0035295 0.00079 1.99814 26.22725 41 96 tube inside development GO: 0009792 0.000941 2.036269 24.04165 38 88 embryonic inside development ending in birth or egg hatching GO: 0006817 0.001021 2.34154 16.39203 28 60 phosphate inside transport GO: 0035270 0.001048 3.649792 7.092567 15 26 endocrine inside system development GO: 0001709 0.001066 3.642225 7.103214 15 26 cell fate inside/promoter determination GO: 0030326 0.001093 3.244364 8.469216 17 31 embryonic limb inside/promoter morphogenesis GO: 0043583 0.00125 2.813623 10.65482 20 39 ear development inside GO: 0051254 0.001323 1.583569 56.00611 76 205 positive inside regulation of RNA metabolic process GO: 0001649 0.00135 3.73849 6.556812 14 24 osteoblast inside differentiation GO: 0016477 0.001648 3.088396 3.84543 11 194 cell migration promoter GO: 0001756 0.001671 5.336521 4.098008 10 15 somitogenesis inside GO: 0030154 0.001739 1.236387 282.3619 323 1062 cell inside differentiation GO: 0009953 0.001796 3.338246 7.376414 15 27 dorsal/ventral inside pattern formation GO: 0007420 0.0018 2.174124 17.64753 29 65 brain inside development GO: 0031325 0.001905 1.420589 91.79537 116 336 positive inside regulation of cellular metabolic process GO: 0031016 0.002219 7.112091 3.005206 8 11 pancreas inside development GO: 0030878 0.002221 15.99486 1.912404 6 7 thyroid gland inside development GO: 0045597 0.002287 2.243075 15.57243 26 57 positive inside regulation of cell differentiation GO: 0042472 0.002303 3.398158 6.830013 14 25 inner ear inside morphogenesis GO: 0001654 0.002407 2.671956 10.38162 19 38 eye inside development GO: 0048534 0.002449 1.718494 34.96967 50 128 hemopoietic or inside lymphoid organ development GO: 0001764 0.002689 2.838035 9.015617 17 33 neuron inside migration GO: 0009653 0.002713 1.700625 35.04919 50 132 anatomical inside structure morphogenesis GO: 0007267 0.002732 1.482421 66.00984 86 245 cell-cell signaling inside GO: 0045944 0.002991 1.705597 34.42327 49 126 positive inside regulation of transcription from RNA polymerase II promoter GO: 0048666 0.003085 4.51368 4.323974 10 16 neuron inside development GO: 0006812 0.003153 1.368558 103.8162 128 380 cation transport inside GO: 0007507 0.003355 2.047192 18.26662 29 67 heart inside development GO: 0003007 0.003369 4.446488 4.371208 10 16 heart inside morphogenesis GO: 0048513 0.003421 1.838106 17.14586 29 865 organ promoter development GO: 0042311 0.003551 11.5716 0.317149 3 16 vasodilation promoter GO: 0030855 0.003746 3.114549 7.103214 14 26 epithelial cell inside differentiation GO: 0045761 0.004025 2.670723 9.288818 17 34 regulation of inside adenylate cyclase activity GO: 0007595 0.004249 10.74396 0.336971 3 17 lactation promoter GO: 0007369 0.004843 3.66869 5.19081 11 19 gastrulation inside GO: 0030902 0.004843 3.66869 5.19081 11 19 hindbrain inside development GO: 0001935 0.005025 10.02667 0.356792 3 18 endothelial cell promoter proliferation GO: 0042136 0.005064 5.333333 3.278406 8 12 neurotransmitter inside biosynthetic process GO: 0048839 0.005371 6.288015 0.713585 4 36 inner ear promoter development GO: 0002052 0.005562 Inf 1.092802 4 4 positive inside regulation of neuroblast proliferation GO: 0009249 0.005562 Inf 1.092802 4 4 protein inside lipoylation GO: 0021871 0.005562 Inf 1.092802 4 4 forebrain inside regionalization GO: 0045885 0.005562 Inf 1.092802 4 4 positive inside regulation of survival gene product activity GO: 0001505 0.005663 1.970186 18.03123 28 66 regulation of inside neurotransmitter levels GO: 0048732 0.005765 2.527244 9.547686 17 35 gland inside development GO: 0006814 0.005976 1.839114 22.12924 33 81 sodium ion inside transport GO: 0006023 0.006192 3.81075 4.644409 10 17 aminoglycan inside biosynthetic process GO: 0042127 0.006293 1.345211 97.53259 119 357 regulation of cell inside proliferation GO: 0030324 0.006442 2.403541 10.38162 18 38 lung inside development GO: 0002062 0.006821 7.996328 2.185604 6 8 chondrocyte inside differentiation GO: 0040018 0.006821 7.996328 2.185604 6 8 positive inside regulation of multicellular organism growth GO: 0007611 0.006957 2.306642 11.20122 19 41 learning and/or inside memory GO: 0045661 0.007037 13.32416 1.639203 5 6 regulation of inside myoblast differentiation GO: 0045773 0.007037 13.32416 1.639203 5 6 positive inside regulation of axon extension GO: 0007154 0.007222 1.475146 52.15117 68 2631 cell promoter communication GO: 0009880 0.007599 2.891016 6.830013 13 25 embryonic inside pattern specification GO: 0030321 0.007689 19.97143 0.138753 2 7 transepithelial promoter chloride transport GO: 0031018 0.007689 19.97143 0.138753 2 7 endocrine promoter pancreas development GO: 0008015 0.007821 3.285874 2.279507 7 115 blood circulation promoter GO: 0000165 0.008109 1.644346 30.87166 43 113 MAPKKK cascade inside GO: 0007215 0.008131 3.260608 5.46401 11 20 glutamate inside signaling pathway GO: 0051056 0.008464 1.474653 50.8153 66 186 regulation of inside small GTPase mediated signal transduction GO: 0007204 0.008553 5.435497 0.812694 4 41 elevation of promoter cytosolic calcium ion concentration GO: 0006936 0.008566 3.225464 2.319151 7 117 muscle promoter contraction GO: 0040011 0.008827 1.964954 16.11883 25 59 locomotion inside GO: 0030900 0.008854 2.292153 10.64347 18 39 forebrain inside development GO: 0031324 0.009247 1.322222 98.62539 119 361 negative inside regulation of cellular metabolic process GO: 0042592 0.009255 1.337753 90.42937 110 331 homeostatic inside process GO: 0002009 0.009685 2.920795 6.26769 12 23 morphogenesis inside of an epithelium GO: 0001656 0.009895 2.910563 6.283612 12 23 metanephros inside development GO: 0042445 0.00997 2.136582 12.29402 20 45 hormone inside metabolic process

TABLE 16 Gene ontology functional categories enriched in hypomethylated C-DMRs (P < 0.01) GOBPID Pvalue OddsRatio ExpCount Count Size Term Region GO: 0032501 7.53E−07 1.311239 504.7175 590 2357 multicellular inside organismal process GO: 0007155 2.18E−06 1.601385 112.8494 157 527 cell adhesion inside GO: 0032502 5.33E−05 1.245694 503.4327 572 2351 developmental inside process GO: 0006816 9.88E−05 2.422182 18.41566 34 86 calcium ion inside transport GO: 0007165 0.000127 1.308432 258.2146 308 1226 signal transduction inside GO: 0009887 0.000132 1.632507 64.02653 91 299 organ inside morphogenesis GO: 0051056 0.000185 1.814148 39.82921 61 186 regulation of small inside GTPase mediated signal transduction GO: 0007399 0.000207 1.405645 134.4771 171 628 nervous system inside development GO: 0048731 0.000231 1.401666 134.76 171 641 system inside development GO: 0006812 0.000257 1.522005 81.37151 110 380 cation transport inside GO: 0007154 0.000309 1.636925 55.3083 79 271 cell communication inside GO: 0006817 0.00031 2.64056 12.84813 25 60 phosphate inside transport GO: 0048667 0.000336 2.058652 23.98318 40 112 neuron inside morphogenesis during differentiation GO: 0007409 0.00043 2.069855 22.69837 38 106 axonogenesis inside GO: 0001525 0.000486 2.076003 22.05596 37 103 angiogenesis inside GO: 0003015 0.000486 3.163871 8.351286 18 39 heart process inside GO: 0032989 0.000504 1.657806 48.60877 70 227 cellular structure inside morphogenesis GO: 0035023 0.000542 2.353129 15.41776 28 72 regulation of Rho inside protein signal transduction GO: 0030879 0.000937 7.363679 2.569627 8 12 mammary gland inside development GO: 0008016 0.001128 3.10623 7.494744 16 35 regulation of heart inside contraction GO: 0006811 0.001488 2.18175 15.51374 27 74 ion transport inside GO: 0031175 0.001662 1.828747 26.55281 41 124 neurite inside development GO: 0006820 0.001971 1.806743 26.76694 41 125 anion transport inside GO: 0048858 0.002059 1.654343 37.47372 54 175 cell projection inside morphogenesis GO: 0050801 0.002091 1.644364 38.33026 55 179 ion homeostasis inside GO: 0001568 0.002126 1.768872 28.48003 43 133 blood vessel inside development GO: 0048661 0.002212 18.39035 1.284813 5 6 positive regulation inside of smooth muscle cell proliferation GO: 0007268 0.002312 1.542972 50.32185 69 235 synaptic inside transmission GO: 0006813 0.002495 1.749208 28.69416 43 134 potassium ion inside transport GO: 0030324 0.00321 2.681625 8.137151 16 38 lung development inside GO: 0050877 0.003314 1.32209 120.3442 147 562 neurological system inside process GO: 0006029 0.003629 10.84488 0.312344 3 30 proteoglycan promoter metabolic process GO: 0051147 0.004404 7.357712 1.92722 6 9 regulation of inside muscle cell differentiation GO: 0008299 0.005816 5.151501 2.569627 7 12 isoprenoid inside biosynthetic process GO: 0007265 0.005855 1.529967 41.75643 57 195 Ras protein signal inside transduction GO: 0005513 0.006372 9.194002 1.498949 5 7 detection of inside calcium ion GO: 0009395 0.006372 9.194002 1.498949 5 7 phospholipid inside catabolic process GO: 0003001 0.007507 2.029051 13.2764 22 62 generation of a inside signal involved in cell-cell signaling GO: 0043062 0.008118 1.973458 14.13295 23 66 extracellular inside structure organization and biogenesis GO: 0007528 0.009022 5.517581 2.141355 6 10 neuromuscular inside junction development GO: 0007613 0.009022 5.517581 2.141355 6 10 memory inside GO: 0045884 0.009022 5.517581 2.141355 6 10 regulation of inside survival gene product expression GO: 0048659 0.009022 5.517581 2.141355 6 10 smooth muscle cell inside proliferation GO: 0007242 0.0098 1.194127 242.8297 274 1134 intracellular inside signaling cascade GO: 0030949 0.009808 Inf 0.642407 3 3 positive regulation inside of vascular endothelial growth factor receptor signaling pathway GO: 0035313 0.009808 Inf 0.642407 3 3 wound healing, inside spreading of epidermal cells GO: 0045662 0.009808 Inf 0.642407 3 3 negative regulation inside of myoblast differentiation GO: 0045740 0.009808 Inf 0.642407 3 3 positive regulation inside of DNA replication

TABLE 17 Regions with tissue-specific differential methylation (T-DMRs) at a FDR of 5% in mus musculus. chromosome start end chr10 126979292 126980671 chr10 127656871 127658217 chr10 24887401 24889329 chr10 28888124 28888716 chr10 33988268 33988975 chr10 53437662 53438762 chr10 67306310 67306619 chr10 76555574 76556749 chr10 79735316 79736395 chr11 100635649 100638591 chr11 102212266 102212933 chr11 115107596 115109335 chr11 115111691 115113355 chr11 120394702 120395960 chr1 134507754 134508470 chr11 3811935 3812582 chr1 157570104 157573766 chr11 57912170 57913262 chr1 158417149 158418321 chr11 58766015 58766738 chr11 59954757 59955965 chr1 162825603 162826433 chr1 162979500 162980205 chr11 63695421 63696067 chr11 70847335 70849134 chr11 78365131 78365880 chr11 79408077 79409420 chr11 81782037 81782297 chr11 83112200 83113213 chr11 84336675 84338372 chr1 186509025 186510266 chr1 186697870 186699731 chr1 193522768 193523343 chr11 98586369 98587025 chr11 98587096 98589090 chr12 108421230 108422853 chr12 113167928 113169389 chr12 30114031 30115188 chr12 30240986 30242545 chr12 31879295 31880075 chr12 53621042 53623143 chr12 55778410 55779489 chr12 58362526 58363068 chr12 60013490 60014134 chr12 70826084 70826557 chr12 70827383 70827994 chr12 71143556 71145601 chr12 71263656 71264508 chr12 73151554 73155106 chr12 83257544 83259350 chr12 99300922 99301356 chr13 49157646 49158486 chr13 52165707 52166249 chr13 55162752 55164518 chr13 55477782 55479287 chr13 58353772 58354380 chr13 58818797 58820212 chr13 70203147 70204256 chr13 70633047 70633499 chr14 102537320 102538579 chr14 104219050 104219694 chr14 20310849 20311379 chr14 24418637 24419041 chr14 30097079 30098320 chr14 50925011 50925826 chr14 53864878 53866395 chr14 54043521 54045533 chr14 54515663 54516328 chr14 64214773 64215654 chr14 84989766 84990308 chr15 100321886 100322823 chr15 101114047 101116123 chr15 102016174 102017697 chr15 102321308 102321977 chr15 27417171 27417674 chr15 27968069 27968486 chr15 39684465 39687107 chr15 68759467 68761004 chr15 68761548 68763148 chr15 78543180 78543923 chr15 79602362 79603288 chr15 82533583 82534176 chr15 85508940 85510281 chr15 85564473 85565120 chr15 89202174 89205204 chr15 91204682 91205405 chr15 96467000 96469097 chr15 96570462 96571826 chr16 24114726 24115655 chr16 32564545 32567256 chr16 42108443 42109471 chr16 85052975 85055043 chr17 12502541 12503011 chr17 27384570 27385268 chr17 28786279 28786821 chr17 34443844 34444281 chr17 34739247 34740179 chr17 56073635 56074076 chr17 63187176 63187802 chr17 73446574 73447083 chr17 73983627 73984097 chr17 80466736 80468474 chr17 84696081 84698211 chr18 25868426 25868929 chr18 34988882 34989739 chr18 36789615 36790328 chr18 37945287 37947408 chr18 38992510 38993187 chr18 60729232 60731679 chr18 61044585 61044998 chr18 65349251 65349859 chr18 75560629 75561096 chr1 91413137 91414588 chr19 25178728 25179405 chr19 34813801 34814130 chr19 44354649 44355473 chr19 45043522 45045155 chr19 46817593 46818555 chr19 54252753 54254455 chr19 55802705 55803957 chr19 5955473 5957745 chr19 8906711 8907458 chr2 104208681 104210622 chr2 130849724 130850548 chr2 163238725 163239474 chr2 179955182 179957788 chr2 33190742 33192554 chr2 65841897 65843222 chr2 93621671 93622925 chr3 121522532 121522894 chr3 121522949 121524010 chr3 121802762 121803376 chr3 127151720 127152797 chr3 135373833 135375158 chr3 83096129 83096809 chr3 83137592 83139735 chr3 87004578 87005432 chr4 117381676 117383055 chr4 125561574 125562182 chr4 136994230 136995186 chr4 138238001 138239488 chr4 148063477 148065006 chr4 150869280 150870206 chr4 154185273 154188604 chr4 35285562 35286638 chr4 42972011 42973852 chr4 42973973 42974614 chr4 59310854 59311432 chr4 98613661 98614914 chr5 107645478 107646164 chr5 113458417 113459346 chr5 115071773 115072276 chr5 124865500 124866267 chr5 143070755 143071267 chr5 143233197 143234288 chr5 65627192 65629135 chr5 90008195 90009248 chr6 101163944 101165305 chr6 119284067 119284606 chr6 124825932 124826609 chr6 147219158 147219979 chr6 38843094 38844092 chr6 55608676 55609270 chr6 83108264 83109323 chr6 83150997 83151731 chr6 83403666 83405735 chr6 85995461 85996279 chr6 90432664 90433797 chr7 113850061 113850390 chr7 121496715 121497551 chr7 127061589 127063103 chr7 127063311 127064541 chr7 130484240 130486315 chr7 139037295 139038319 chr7 140624014 140624754 chr7 140757459 140759046 chr7 15951594 15952451 chr7 18081819 18082463 chr7 24596110 24596727 chr7 24954217 24954966 chr7 26968540 26971209 chr7 27442446 27443207 chr7 29225581 29226432 chr7 30151377 30151814 chr7 30822453 30823520 chr7 45652166 45653161 chr7 49647288 49647932 chr7 55711682 55712182 chr7 79389572 79390003 chr7 96825159 96825599 chr8 111425321 111427570 chr8 111588936 111589513 chr8 111633592 111634305 chr8 112137088 112141065 chr8 112859398 112860273 chr8 124619935 124620738 chr8 4257372 4258328 chr8 47706238 47707539 chr8 48784788 48786354 chr8 87673614 87674328 chr8 87782082 87783422 chr9 106313871 106314629 chr9 106534209 106534400 chr9 108047150 108047903 chr9 108453798 108454337 chr9 110094621 110095928 chr9 110100907 110102298 chr9 114690009 114691953 chr9 21385742 21386110 chr9 36584091 36585669 chr9 44627976 44630076 chr9 45183701 45184379 chr9 64820556 64821893 chr9 66150149 66150724 chr9 69092444 69093502 chr9 83050145 83051215 chr9 95362984 95365053 chr9 99241977 99242273 chr9 99242400 99245369 chrX 147681816 147682532 chr10 28888088 28888716 chr10 53437662 53438762 chr10 80900473 80901327 chr1 108000968 108002063 chr11 102212266 102212933 chr11 109235707 109237233 chr11 22662409 22662984 chr11 3811935 3812546 chr11 53612110 53613003 chr1 158417149 158418321 chr11 58765982 58766738 chr1 162979533 162980205 chr1 166291190 166292146 chr11 69841848 69842984 chr11 70847302 70848750 chr11 83464295 83464906 chr1 186430240 186431931 chr1 186697939 186699731 chr1 187102071 187102469 chr11 94938664 94939659 chr11 96105595 96107038 chr11 96133733 96134386 chr11 96143071 96143511 chr11 97613914 97614906 chr11 98587132 98589159 chr12 111186780 111187702 chr12 30241655 30242545 chr12 45197481 45198904 chr12 53621042 53623212 chr12 55778410 55779555 chr12 70827419 70827994 chr12 71263656 71264541 chr12 73151554 73155106 chr12 83257544 83259284 chr12 99300922 99301392 chr13 21513212 21513649 chr13 35745963 35748082 chr13 49157646 49158525 chr13 52540971 52542658 chr13 55477782 55479254 chr13 58353808 58354380 chr13 58818728 58820212 chr14 104219050 104219694 chr1 41549409 41549774 chr14 19166845 19167417 chr14 30097112 30098248 chr14 51166853 51167672 chr14 53733790 53734791 chr14 53865226 53866395 chr14 54043521 54045602 chr14 63546684 63549154 chr14 64214773 64215754 chr14 65819186 65820690 chr14 7008650 7010062 chr15 100321886 100322856 chr15 27417132 27417674 chr15 27968069 27968486 chr15 37079209 37080133 chr15 39490076 39490510 chr1 55352211 55352609 chr15 68759313 68760932 chr15 79919013 79919906 chr15 85508940 85509726 chr15 97252471 97253256 chr16 42108443 42109435 chr16 57391719 57392402 chr16 58352052 58352837 chr1 66402075 66402928 chr16 81084948 81085859 chr16 85051940 85055848 chr17 34443844 34444281 chr17 5451100 5452848 chr1 77394488 77395317 chr18 25868426 25868929 chr18 32701107 32702376 chr18 34988219 34989670 chr18 37945468 37947408 chr18 60745072 60745914 chr18 61044585 61044998 chr18 69685663 69686202 chr1 91765466 91766315 chr19 43658075 43659637 chr19 5117032 5117184 chr19 55802945 55803713 chr19 58130598 58131158 chr2 113628540 113629780 chr2 148368993 148369534 chr2 148369553 148369951 chr2 179955743 179957821 chr2 92232293 92234278 chr2 93621635 93622925 chr3 127151720 127152764 chr3 135374094 135375194 chr3 136611345 136612055 chr3 138741405 138741737 chr3 144130403 144131566 chr3 28784766 28785098 chr3 87004578 87005465 chr4 114409124 114411976 chr4 125561505 125562182 chr4 133522280 133523272 chr4 135299619 135300935 chr4 148149542 148150642 chr4 154186710 154188227 chr4 41756406 41756948 chr5 113546929 113547504 chr5 116169864 116171999 chr5 143070791 143071297 chr5 52679340 52680512 chr6 119284067 119284606 chr6 145003894 145005549 chr6 147219194 147220132 chr6 52105770 52106804 chr6 52109489 52110473 chr6 52118616 52120690 chr6 52130359 52133065 chr6 52134341 52135335 chr6 55608676 55609444 chr6 88170526 88171206 chr6 88839352 88840134 chr7 121496679 121497518 chr7 130484240 130486315 chr7 18081786 18082427 chr7 26968690 26971209 chr7 44735606 44736776 chr7 44919263 44920252 chr7 45652130 45653257 chr7 62297270 62297671 chr7 79389605 79390003 chr7 96825159 96825491 chr8 108238955 108239740 chr8 111425321 111427570 chr8 112137124 112138948 chr8 112139103 112141065 chr8 122007273 122007887 chr8 124535330 124537432 chr8 124620001 124620738 chr8 127546771 127548576 chr8 14113008 14113968 chr8 60218064 60218813 chr9 106313871 106314527 chr9 108119711 108121117 chr9 108453798 108454337 chr9 21385742 21386077 chr9 45183767 45185397 chr9 66150221 66150724 chr9 72490554 72492061 chr9 83601188 83601727 chr9 89629425 89629895 chr10 126979364 126980638 chr10 127020412 127022913 chr10 127656943 127658217 chr10 41890728 41891096 chr10 80692225 80693889 chr1 108001004 108001925 chr11 103160706 103161842 chr11 109293793 109294485 chr11 115111730 115113316 chr11 120237470 120237940 chr11 120611069 120611503 chr11 120766874 120767062 chr11 22662409 22662984 chr1 135191990 135192493 chr1 139952104 139952436 chr1 163084626 163084958 chr11 69180232 69180594 chr11 69180685 69181225 chr11 78365131 78365880 chr1 193522768 193523343 chr12 113168003 113169353 chr12 118743027 118744363 chr12 31879325 31880075 chr12 58362526 58363068 chr12 60013490 60014101 chr12 71144570 71145463 chr12 84942362 84943216 chr12 86635810 86637096 chr12 87778503 87779744 chr13 40897757 40899363 chr13 52165707 52166249 chr13 52987856 52990349 chr13 92215958 92216461 chr14 120387491 120388301 chr14 46395955 46396851 chr14 50829932 50832484 chr14 50832626 50833966 chr14 56055810 56057340 chr15 102015649 102016017 chr15 10928320 10928886 chr15 37079176 37080133 chr15 76133841 76134551 chr15 78236579 78237388 chr15 85564542 85565081 chr15 88692381 88692779 chr15 89202174 89205168 chr15 89384916 89385488 chr15 97252471 97253256 chr16 24114972 24115619 chr16 57391719 57392402 chr1 66935142 66935648 chr16 84715521 84716705 chr16 92895397 92895810 chr17 12502580 12503011 chr17 24422788 24423243 chr17 28786279 28786785 chr17 45349645 45350289 chr17 80466736 80468438 chr17 84696258 84698178 chr18 36789687 36790328 chr18 60729268 60731718 chr18 60732259 60732978 chr18 65349251 65349859 chr18 70653550 70654152 chr18 75548120 75548380 chr19 25178692 25179405 chr19 29115205 29115927 chr19 34813837 34814130 chr19 34942556 34943841 chr19 37238481 37239098 chr19 37328982 37330121 chr19 43654651 43655382 chr1 94901325 94902392 chr19 54252753 54254134 chr19 5955777 5957547 chr2 163238827 163239474 chr2 30028984 30029874 chr2 58379608 58380903 chr2 58381061 58381699 chr3 121523021 121523974 chr3 121894506 121894976 chr3 83096129 83096809 chr3 83137592 83139768 chr4 105959139 105959501 chr4 105959592 105960239 chr4 135411138 135412272 chr4 138238949 138239419 chr4 140403814 140404461 chr4 148149575 148150570 chr4 148628253 148628720 chr4 149697690 149698580 chr4 150869421 150870134 chr4 35285775 35286638 chr4 40456117 40456761 chr4 59310821 59311432 chr4 97328719 97329291 chr5 113197288 113198073 chr5 113458495 113459346 chr5 116706401 116708027 chr5 118239146 118240513 chr5 123395820 123396041 chr5 124865500 124866234 chr5 65627192 65629135 chr6 119382061 119382708 chr6 124629874 124631263 chr6 31119916 31120566 chr6 85903153 85903656 chr6 88156373 88156597 chr7 127061589 127063136 chr7 132958148 132959237 chr7 140624014 140624754 chr7 140757459 140757896 chr7 15951594 15952415 chr7 29942884 29943006 chr7 30662992 30664459 chr7 49211159 49211842

TABLE 18 Regions with consistent cancer-specific differential methylation (C-DMRs) at a FDR of 5%. TSS chr start end deltaM fdr gene name relation distance CGI distToCGI chr7 153219537 153220325 −1.02674 0.00000131 DPP6 inside 1096601 Far −2938 chr21 37858785 37859555 −1.08401 0.00000164 DYRK1A upstream 49238 Shore −369 chr11 117907224 117907958 1.00157 0.00000328 TMEM25 inside 3800 Shore 0 chr19 5159412 5160095 −1.02228 0.00000628 PTPRS inside 131718 Shore 1469 chr11 65947037 65948698 −0.88203 0.00000638 NPAS4 inside 2054 Shore −1064 chr22 35335058 35337265 −0.87143 0.00000665 CACNG2 inside 91583 Far −44058 chr19 60702504 60703541 −0.94123 0.00000892 NAT14 upstream 11758 Far 3082 chrX 135941512 135943110 0.904877 0.0000093 GPR101 promoter 14 Island 0 chr6 52637426 52638797 −0.88756 0.000013 TMEM14A downstream 20544 Shore 0 chr7 27106893 27108203 0.870706 0.0000156 HOXA2 inside 715 Shore 1503 chr5 1718233 1720117 −0.85903 0.0000205 MRPL36 downstream 132828 Shore 70 chr19 5297115 5297780 −0.93292 0.0000226 PTPRS upstream 5302 Far −5054 chr2 147061970 147063253 −0.86015 0.0000229 ACVR2A downstream 1341608 Shore −121 chr8 819298 820365 −0.87447 0.0000243 ERICH1 upstream 148073 Shore 1879 chr22 48414225 48414719 −0.96407 0.0000249 C22orf34 inside 22470 Shore −1806 chr13 113615559 113616275 −0.91383 0.000027 FAM70B inside 35597 cover 0 chr15 58474612 58476390 −0.81855 0.0000399 ANXA2 inside 1086 Shore 651 chr7 3984567 3985861 −0.84076 0.0000663 SDK1 inside 285898 Far 134310 chr20 33650044 33650409 0.946503 0.0000695 SPAG4 downstream 21969 Shore 1724 chr13 24843229 24844069 0.886019 0.0000738 ATP8A2 downstream 649349 Shore 0 chr4 62618513 62619190 −0.87267 0.0000761 LPHN3 inside 1571 Far −552670 chr3 193607217 193607552 −0.93573 0.0001116 FGF12 inside 1153 Shore 960 chr10 5774977 5775480 0.88404 0.00012066 ASB13 upstream 26431 Far −7274 chr3 82629728 82630444 −0.83773 0.00014135 GBE1 upstream 736294 Far 309227 chr15 70198455 70199761 −0.825 0.00014832 SENP8 inside 20595 Shore −399 chr8 144543158 144544971 −0.7704 0.00017229 RHPN1 upstream 7196 Far −9061 chr19 63146186 63149398 −0.7009 0.00020946 ZNF256 inside 1490 Shore 1100 chr7 27121979 27122917 0.793013 0.00021477 HOXA3 inside 2821 Shore −28 chr10 129973472 129974329 −0.79104 0.00024398 MKI67 upstream 158828 Far −19204 chr5 175019374 175020470 −0.78658 0.00032057 HRH2 downstream 23691 Shore −1012 chr15 90735153 90736013 −0.77464 0.0003365 ST8SIA2 downstream 76946 Shore 1668 chr20 15408602 15408931 −0.87712 0.00036397 C20orf133 inside 572907 Far 1092905 chr12 128898887 128900474 −0.72032 0.00038766 TMEM132D inside 53691 Far 5162 chr20 56858718 56860982 0.659681 0.00039705 GNAS inside 58661 cover 0 chr8 118032349 118033443 −0.77052 0.00039943 LOC441376 upstream 7947 Far −12209 chr19 55631492 55632172 −0.78998 0.00040909 MYBPC2 inside 29216 Far −4003 chr7 27151538 27154015 0.661581 0.00040909 HOXA6 covers 0 Shore 0 chr22 36144969 36145868 0.759849 0.00041153 LRRC62 upstream 43445 Shore 0 chr20 16499527 16500276 −0.77576 0.00043547 C20orf23 inside 1801 Shore 1560 chr7 4865828 4866751 −0.75229 0.00043806 PAPOLB inside 1399 Shore 1111 chr8 17060922 17062917 −0.68442 0.0005137 ZDHHC2 inside 61694 Shore −1761 chr12 108883476 108884015 −0.79921 0.00055912 GIT2 inside 34467 Far 34165 chr1 14092469 14093393 0.743518 0.00056075 PRDM2 upstream 68308 Shore −145 chr9 101171277 101172098 −0.75185 0.00056896 SEC61B upstream 138557 Far −72243 chr7 27129188 27131713 0.62826 0.00063513 HOXA3 inside 1450 cover 0 chr9 94987821 94990808 0.598843 0.00069825 WNK2 inside 131865 Shore −165 chr7 145026589 145027407 −0.73801 0.00073506 CNTNAP2 downstream 2721611 Far 416556 chr5 16236219 16237106 −0.72798 0.00074772 FBXL7 upstream 243320 Far −2799 chr6 168586102 168588777 0.621865 0.00078025 SMOC2 inside 221818 Shore −153 chr7 27147025 27148381 0.688088 0.00079587 HOXA5 overlaps 3′ 1430 Shore 757 chr6 50873235 50873811 −0.77277 0.00080038 TFAP2B downstream 45822 Far 21434 chr9 136758751 136759638 −0.72195 0.000835 COL5A1 inside 116868 Far 40696 chr17 74692733 74693892 −0.71657 0.00085162 LOC146713 downstream 237673 Shore −909 chr20 44094989 44097479 −0.68354 0.00086367 SLC12A5 inside 24716 Shore −634 chr12 128900559 128905004 −0.58154 0.00089325 TMEM132D inside 49161 Shore 632 chr9 137437549 137438193 −0.74586 0.00103239 KIAA0649 downstream 82366 Far 6096 chr8 1032932 1034329 −0.68718 0.00109097 ERICH1 upstream 361707 Shore 1637 chr20 56841054 56842229 −0.67616 0.00110609 GNAS downstream 77414 Far 5761 chr11 2190993 2192468 −0.65402 0.00113066 TH upstream 41383 Far −46172 chr7 80386676 80389252 −0.60191 0.00118778 SEMA3C promoter 74 Shore −14 chr8 846979 849372 −0.59665 0.00119103 ERICH1 upstream 175754 Far −6574 chr3 191521264 191521946 −0.73631 0.00122064 CLDN1 inside 962 Shore 562 chr3 193338227 193339771 −0.67305 0.00123738 FGF12 downstream 268934 Far 268741 chr16 86201868 86205260 −0.67852 0.00127148 JPH3 inside 83999 Shore 327 chr22 48392638 48393144 −0.76072 0.00128536 C22orf34 downstream 44045 Shore 1818 chr8 966339 968246 −0.6265 0.00129586 ERICH1 upstream 295114 Shore −581 chr11 73980533 73981178 −0.78485 0.00133142 POLD3 downstream 50234 Shore 0 chr18 32022373 32023451 −0.68242 0.00135317 MOCOS inside 79230 Shore −230 chr18 73809395 73809862 −0.76199 0.00144347 GALR1 upstream 698315 Far 9620 chr13 113548528 113549312 0.70947 0.00145514 GAS6 inside 41083 Shore −393 chr3 129687744 129688211 0.759315 0.00151474 GATA2 inside 6506 Shore 0 chr14 95577239 95578964 −0.6455 0.00151879 C14orf132 inside 50921 Shore −1084 chr7 92075312 92075740 −0.77713 0.00152285 CDK6 inside 225407 Far −17503 chr12 16648850 16649638 0.700737 0.00155156 LMO3 inside 1059 Far −693049 chr19 43436533 43438474 0.71493 0.00159765 PPP1R14A inside 537 Shore 4 chr7 50436490 50438081 −0.63917 0.00161044 IKZF1 overlaps 5′ 0 Shore −596 chr13 42886712 42887185 −0.75536 0.00161901 PIG38 inside 252469 Far 370673 chr8 100031420 100033208 0.611014 0.00162763 OSR2 inside 291 Shore −806 chr16 31139986 31140921 −0.70124 0.00167576 TRIM72 inside 3089 Far 2105 chr13 87123702 87125116 0.642127 0.00169358 SLITRK5 inside 4752 cover 0 chr2 172826386 172826961 −0.72901 0.00172973 DLX2 upstream 150663 Far −18042 chr12 88272781 88274261 −0.65038 0.00193625 DUSP6 promoter 2355 Shore −506 chr7 50103520 50103993 −0.74337 0.00198218 ZPBP promoter 149 Shore −53 chr7 1182527 1183550 −0.70109 0.00201859 ZFAND2A upstream 16204 Far −15857 chrX 23042002 23042541 −0.75918 0.00206093 DDX53 upstream 111878 Far 217664 chr8 98356982 98358151 −0.64161 0.00212045 TSPYL5 inside 1200 Shore 629 chr11 132451645 132454794 −0.55186 0.00212594 OPCML inside 452818 Far 2108 chr4 172202321 172202941 −0.71014 0.00212594 GALNT17 downstream 1996342 Far 767368 chr11 133791126 133793856 −0.56461 0.00224415 B3GAT1 upstream 4105 Far −2997 chr7 129912706 129913314 −0.69892 0.00255614 MEST overlaps 3′ 20050 Shore 0 chr12 38783104 38784488 0.607732 0.00260186 SLC2A13 inside 1439 Shore 741 chr13 112142200 112143003 −0.6783 0.00268194 C13orf28 upstream 5199 Far 11335 chr7 90064351 90065026 0.683928 0.00268194 PFTK1 downstream 612813 Shore −51 chr11 103540808 103541706 −0.6721 0.0027228 PDGFD promoter 572 Shore −540 chr21 42057698 42059424 −0.5888 0.00274344 RIPK4 inside 893 Shore 0 chr2 4028568 4028969 −0.73928 0.00285588 ALLC upstream 300436 Island 0 chr11 2243752 2244711 −0.64164 0.00286305 ASCL2 downstream 4046 Shore 1969 chr5 11954677 11955633 −0.64541 0.00287742 CTNND2 inside 1476 Shore 917 chr5 7900127 7901165 −0.66262 0.00292092 FASTKD3 downstream 20949 Shore 1780 chr13 109229281 109231903 −0.54967 0.00303226 IRS2 inside 5011 Shore 564 chr13 110126656 110127336 0.674417 0.00312403 FLJ12118 inside 29127 Shore 1831 chr20 48778557 48780262 −0.59546 0.00331501 PARD6B downstream 23421 Shore 510 chr14 100997052 100997806 −0.66962 0.00337263 DIO3 downstream 101733 Shore −1304 chr3 28591265 28591843 0.687621 0.00340596 ZCWPW2 upstream 49632 Shore 0 chr11 133445524 133446466 −0.65981 0.0036033 JAM3 inside 80392 Shore −633 chr5 132392 134453 −0.57597 0.00364751 KIAA1909 downstream 108624 Shore 1975 chrX 142544143 142544478 −0.74381 0.0037556 SLITRK4 inside 6206 Far 4598 chr20 4928754 4929383 0.695671 0.00377388 SLC23A2 inside 761 Far 111977 chr12 112557273 112558104 −0.69254 0.00396112 LHX5 upstream 163014 Far −43138 chr16 85092993 85096638 0.487544 0.0039899 FOXF1 downstream 8931 Shore 0 chr10 1437553 1439129 −0.58557 0.00404802 ADARB2 inside 330540 Far −7539 chr2 242632887 242634574 −0.66358 0.00405778 FLJ33590 upstream 168241 Shore 1740 chr2 4028054 4028513 −0.72737 0.00406756 ALLC upstream 299922 Shore 11 chr19 35406489 35407329 0.646091 0.00410688 ZNF536 downstream 333475 Shore 60 chr7 42233455 42234204 0.645604 0.0041366 GLI3 upstream 4036 Shore 0 chr3 174341049 174341237 −0.80111 0.00415651 SPATA16 inside 457 Far 254724 chr5 3586182 3587073 −0.70608 0.00415651 IRX1 downstream 67442 Shore 1557 chr8 22469866 22470840 −0.62573 0.00420667 SORBS3 inside 18110 Far −4286 chr6 10501022 10502013 0.654216 0.00428802 TFAP2A downstream 18579 Shore −1938 chr11 31966319 31967492 −0.63749 0.00433957 RCN1 downstream 116160 Shore −699 chr1 206062462 206063805 0.576185 0.00447616 LOC148696 overlaps 5′ 0 Far 44680 chr22 47267034 47267870 −0.63064 0.00447616 FAM19A5 downstream 265877 Shore −1327 chr6 80711271 80712497 −0.59439 0.00450821 ELOVL4 inside 1443 Shore 966 chr22 47455137 47456387 −0.58401 0.00455126 FAM19A5 inside 77360 Far −4801 chr11 134186796 134187368 −0.6669 0.00469367 B3GAT1 upstream 399775 Far −48599 chr19 13474169 13475470 −0.62291 0.00469367 CACNA1A inside 2846 Far 2282 chr16 25608094 25609031 −0.61772 0.00474948 HS3ST4 downstream 447477 Shore 1425 chr8 117611158 117611838 −0.64748 0.00474948 EIF3S3 downstream 225404 Far 225273 chr20 3603573 3604604 −0.59867 0.00496688 ADAM33 inside 6133 Shore −800 chr20 59905352 59906092 −0.67681 0.00503732 CDH4 inside 39601 Shore −1622 chr14 52326048 52327382 −0.59637 0.00514462 GNPNAT1 inside 750 Shore 32 chr22 46984389 46985561 −0.58562 0.00514462 LOC388915 upstream 66632 Shore −218 chr7 152249022 152250754 −0.63648 0.00515666 ACTR3B upstream 65627 Far 2095 chr7 27108458 27111260 0.508493 0.00515666 HOXA2 overlaps 5′ 0 cover 0 chr5 158465126 158466712 0.548688 0.00532789 EBF1 upstream 5780 cover 0 chr7 149667109 149668386 0.570024 0.00536522 RARRES2 inside 1252 Shore 6 chr10 3499370 3500060 −0.64479 0.00540278 PITRM1 upstream 294368 Far −8794 chr11 31780765 31782111 0.582076 0.00542795 PAX6 inside 7322 Shore 208 chr7 139121471 139122253 0.621564 0.00552964 TBXAS1 downstream 244217 Shore 1622 chr17 14831980 14832519 −0.66274 0.00555532 FLJ45831 upstream 207736 Far 272136 chr3 148446946 148448043 −0.61085 0.00563299 ZIC4 downstream 159053 Far 111759 chr18 11140361 11141025 −0.64161 0.00567218 FAM38B upstream 452548 Shore −425 chr19 4505154 4505834 −0.63538 0.00569843 SEMA6B inside 3668 Shore 1102 chr4 81341228 81342411 0.593058 0.0057248 PRDM8 inside 2092 Shore 121 chr13 100427954 100428562 −0.65947 0.00583131 VGCNL1 downstream 438251 Far −302230 chr1 221053963 221054618 −0.65816 0.00595319 FLJ43505 upstream 63194 Shore 249 chr9 97304533 97306772 0.538529 0.00598058 PTCH1 inside 3879 Shore 1524 chr10 134597978 134600218 −0.58526 0.00606339 C10orf93 inside 5835 Far 5435 chr10 24025367 24026498 −0.60529 0.00611915 C10orf67 upstream 351590 Shore −383 chr19 50669649 50670313 0.642613 0.00611915 FOSB overlaps 5′ 0 Shore −1547 chr18 491107 491721 0.648047 0.006232 COLEC12 promoter 423 Shore −385 chr17 41333049 41334735 −0.54819 0.00626049 MAPT inside 126808 Far −2213 chr12 52430235 52431320 0.598153 0.00636111 CALCOCO1 upstream 22750 Island 0 chr13 67580738 67581346 −0.63959 0.00640466 PCDH9 upstream 878275 Far 875559 chr5 134553843 134554689 0.606657 0.00641924 H2AFY downstream 208137 Shore 137 chr12 122813965 122816002 −0.551 0.00652206 ATP6V0A2 upstream 3575 Shore −758 chr20 24399131 24400197 0.576626 0.00652206 C20orf39 inside 194969 Island 0 chr2 100303439 100304155 0.61944 0.00664131 LONRF2 upstream 11041 Shore 56 chr13 21140646 21141533 0.594077 0.0067472 FGF9 downstream 32650 Shore 0 chr8 132984463 132985363 −0.6234 0.00679303 KIAA0143 downstream 109587 Shore 141 chr11 1861791 1862726 −0.59254 0.006901 LSP1 inside 7341 Shore −1881 chr10 101273278 101274941 0.562231 0.00691655 NKX2-3 downstream 11326 Shore −348 chr13 113850844 113851732 0.592061 0.00694773 RASA3 inside 64464 Far 2436 chr7 32076356 32076652 0.715435 0.00699473 PDE1C inside 863 Shore 0 chr11 45645190 45645996 −0.6053 0.00701046 CHST1 promoter 1443 Shore −1119 chr19 16876450 16876750 −0.74637 0.00701046 CPAMD8 inside 121718 Far −6593 chr8 53636408 53637226 −0.60042 0.00702621 UNQ9433 inside 3338 Far 2871 chr15 24571127 24572559 −0.55445 0.00705782 GABRB3 promoter 1108 Shore −1091 chr6 133605067 133606789 0.529372 0.00708955 EYA4 inside 287568 Shore 0 chr12 88266873 88268978 −0.49621 0.0072177 DUSP6 inside 1448 Shore 321 chr17 782607 784014 −0.56251 0.00723386 NXN inside 45745 Far 12943 chr19 36535076 36535822 0.607202 0.00734784 TSHZ3 upstream 73062 Shore 0 chr13 20184925 20186542 −0.53392 0.00741367 IL17D inside 8693 Far 7069 chr17 22902924 22903499 −0.63407 0.00763111 KSR1 inside 71344 Far 53125 chr1 32992405 32992989 0.632486 0.00780208 KIAA1522 inside 20168 Shore 0 chr15 32835765 32836849 −0.56319 0.00792371 CX36 promoter 1785 Shore −993 chr12 130718351 130718755 −0.68171 0.00801157 SFRS8 downstream 131479 Far −5588 chr13 36392772 36394298 0.523296 0.00801157 ALG5 downstream 77178 Shore −20 chr20 61186047 61186916 −0.58658 0.00801157 BHLHB4 upstream 77261 Shore 1296 chr11 19323912 19324590 0.644909 0.00802925 E2F8 upstream 104830 inside 0 chr12 111302662 111303588 0.576207 0.00820783 RPL6 downstream 28237 Island 0 chr15 97011564 97012941 0.531506 0.0082802 IGF1R inside 306092 Shore 0 chr16 49141667 49142498 −0.59792 0.00837144 NKD1 inside 83643 Shore −701 chr14 57669356 57669957 −0.63311 0.00853784 C14orf37 inside 18642 Far 18087 chr8 144372893 144373912 −0.59142 0.0085565 LOC338328 promoter 2476 Far 9171 chr9 124022303 124023189 0.590922 0.00861269 LHX6 inside 7615 Shore 0 chr19 3240846 3241458 −0.61842 0.0086692 BRUNOL5 inside 6612 Far 2809 chr16 4101996 4102769 0.594061 0.00884063 ADCY9 inside 3417 Far 2040 chr12 4889938 4890628 0.61045 0.00889841 KCNA1 close to 3′ 1662 inside 0 chr3 151719593 151721472 −0.52881 0.00895651 SERP1 downstream 25645 Far 24824 chr19 41058856 41059386 −0.6366 0.00903448 APLP1 inside 3151 Shore −1192 chr7 71437052 71438143 −0.55387 0.00903448 CALN1 inside 1752 Shore 550 chr9 37023001 37023958 0.572428 0.00919216 PAX5 inside 517 Shore 177 chr20 33336228 33337040 −0.58612 0.00921203 FAM83C overlaps 3′ 6598 Shore −75 chr7 5360426 5361424 0.575878 0.00931195 SLC29A4 upstream 50199 Shore 974 chr18 75366596 75367142 −0.77008 0.00935217 NFATC1 inside 23167 Far 4639 chr6 7413675 7414250 −0.61922 0.00939254 RIOK1 upstream 50409 Far 72267 chr11 64244495 64245421 −0.57059 0.00943306 NRXN2 inside 1814 Shore 1648 chr16 7291114 7292175 −0.5578 0.00949411 A2BP1 inside 408671 Far 2159 chr8 3257254 3257862 −0.61199 0.00949411 CSMD1 inside 1581873 Far −389899 chr5 88221043 88221995 0.573951 0.00955551 MEF2C upstream 6264 Island 0 chr13 99440030 99441015 0.573722 0.00959662 ZIC2 upstream 3012 Shore 0 chr10 102811316 102812341 −0.55274 0.00965857 KAZALD1 overlaps 3′ 2996 inside 0 chr17 71291527 71292204 −0.59686 0.00988864 H3F3B upstream 4073 Shore 237 chr10 112248574 112249464 0.566331 0.00999474 DUSP5 inside 11825 Shore 0 DeltaM is cancer minus normal. FDR is false discovery rate. Columns are chromosome, start, end, delta M, fdr, gene, relation to gene, distance to TSS, relation to CGI, distance to CGI 

1. A method of detecting a cell proliferative disorder comprising: comparing the methylation status of one or more nucleic acid sequences in a sample from a subject suspected of having the disorder, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, to the methylation status of the one or more nucleic acid sequences in a sample from a corresponding normal tissue or individual not having a cell proliferative disorder, wherein an alteration in methylation status is indicative of a cell proliferative disorder.
 2. The method of claim 1, wherein the cell proliferative disorder is cancer.
 3. The method of claim 2, wherein the cancer is colorectal cancer.
 4. The method of claim 1, wherein the nucleic acid sequence is within a gene.
 5. The method of claim 1, wherein the nucleic acid sequence is upstream or downstream of a gene.
 6. The method of claim 1, wherein the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof.
 7. The method of claim 6, wherein the alteration in methylation status is hypomethylation.
 8. The method of claim 6, wherein the alteration in methylation status is hypermethylation.
 9. The method according to claim 1, wherein the methylation status is performed by one or more techniques selected from the group consisting of a nucleic acid amplification, polymerase chain reaction (PCR), methylation specific PCR, bisulfite pyrosequenceing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, microarray technology, and proteomics.
 10. The method of claim 1, wherein the sample is derived from human blood, plasma, serum, lymph, nerve-cell containing tissue, cerebrospinal fluid, biopsy material, tumor tissue, bone marrow, nervous tissue, skin, hair, tears, fetal material, amniocentesis material, uterine tissue, saliva, feces, or sperm.
 11. A plurality of nucleic acid sequences, wherein the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in cancer.
 12. The plurality of claim 11, wherein the nucleic acid sequences are selected from the group consisting of the DMR sequences as set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, and the RARES2 gene.
 13. The plurality of claim 11, wherein the plurality is a microarray.
 14. A plurality of nucleic acid sequences, wherein the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, and wherein the nucleic acid sequences are differentially methylated in tissues derived from the three different embryonic lineages.
 15. The plurality of claim 14, wherein the nucleic acid sequences are selected from the sequences as set forth in FIG. 5 a, FIG. 5 b, Tables 1, 2, 4, 7, 9, 10, 12-14, and
 17. 16. The plurality of claim 14, wherein the plurality is a microarray.
 17. A method of determining clinical outcome, comprising: comparing the methylation status of one or more nucleic acid sequences in a sample from a subject prior to undergoing a therapeutic regimen for a disease or disorder, wherein the disease or disorder is associated with altered methylation of the one or more nucleic acid sequences, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island, to the methylation status of the one or more nucleic acid sequences in a sample from the individual after the therapeutic regimen has been initiated, wherein change in methylation status is indicative a positive clinical outcome.
 18. The method of claim 17, wherein the cell disease or disorder is cancer.
 19. The method of claim 17, wherein the one or more nucleic acid sequence is selected from the group consisting of the DMR sequences as set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, and the RARES2 gene.
 20. The method of claim 17, wherein the altered methylation status is hypomethylation.
 21. The method of claim 17, wherein the altered methylation status is hypermethylation.
 22. A method for providing a methylation map of a region of genomic DNA, comprising: performing comprehensive high-through array-based relative methylation (CHARM) analysis on a sample of labeled, digested genomic DNA.
 23. The method of claim 22, further comprising bisulfite pyrosequencing of the genomic DNA.
 24. A method for detecting a methylation status profile of the nucleic acid of a cancer cell from a tumor or biological sample, comprising: a) hybridizing labeled and digested nucleic acid of a cancer cell from a tumor or biological sample to a DNA microarray comprising at least 2000 nucleic acid sequences, with the proviso that the nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island; b) determining a pattern of methylation from the hybridizing of step a), thereby detecting a methylation profile.
 25. The method of claim 24, further comprising comparing the methylation profile to a methylation profile from hybridization of the microarray with labeled and digested nucleic acid from control cells.
 26. The method of claim 24, wherein the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof.
 27. A detection method for prognosis of a cancer in a subject known to have or suspected of having a cancer associated with altered methylation of one or more nucleic acid sequences, comprising: a) obtaining a tissue sample or bodily fluid containing nucleic acid from a subject; and b) assaying the methylation status of one or more nucleic acid sequences, with the proviso that the one or more nucleic acid sequences are outside of a promoter region of a gene and outside of a CpG island, and wherein the nucleic acid sequence is up to about 2 kb in distance from a CpG island bodily fluid; wherein the presence of altered methylation in the sample from the subject, relative to a corresponding sample from a normal sample, is indicative that the subject is a good for therapy of cancer.
 28. The method of claim 27, wherein the one or more nucleic acid sequence is selected from the group consisting of the DMRs set forth in Tables 1-4, 6, 7, 9, 11, 14-16, 18, the DPP6 gene, the MRPL36 gene, the MEST gene, the GATA-2 gene, the RARES2 gene, and any combination thereof. 